Biomedical subjects

S Nakada

Publications and source records attributed to S Nakada.

At least 37 records · Page 2Linked to original sources

PubMed1998

Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, 'dumbbell RNA/DNA chimeric oligonucleotides', consisting of a sense RNA sequence and its complementary antisense DNA sequence, with two hairpin loop structures. The reaction of the nicked (NDRDON) and circular (CDRDON) dumbbell RNA/ DNA chimeric oligonucleotides with RNase H gave the corresponding antisense phosphodiester oligodeoxynucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target RNA, which gave RNA cleavage products by treatment with RNase H. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligonucleotide (anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide. The CDRDON with four target sites (influenza virus A RNA polymerases (PB1, PB2, PA) and nucleoprotein (NP)) was synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The circular dumbbell DNA/ RNA chimeric oligonucleotide (CDRDON-PB2-as) containing an AUG initiation codon sequence as the target of PB2 showed highly inhibitory effects.

DNA↗

PubMed1998

Regulation of Rad51 function by c-Abl in response to DNA damage.

The Rad51 protein, a homolog of bacterial RecA, functions in DNA double-strand break repair and genetic recombination. Whereas Rad51 catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules, regulation of this function is unknown. The c-Abl tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents. Here we demonstrate that c-Abl interacts constitutively with Rad51. We show that c-Abl phosphorylates Rad51 on Tyr-54 in vitro. The results also show that treatment of cells with ionizing radiation induces c-Abl-dependent phosphorylation of Rad51. Phosphorylation of Rad51 by c-Abl inhibits the binding of Rad51 to DNA and the function of Rad51 in ATP-dependent DNA strand exchange reactions. These findings represent the first demonstration that Rad51 is regulated by phosphorylation and support a functional role for c-Abl in regulating Rad51-dependent recombination in the response to DNA damage.

Cell Line↗

PubMed1998

Surrogate thrombopoietin.

The extracellular domain of human c-Mpl, the receptor for thrombopoietin (TPO), was expressed as a chimeric protein with the interleukin-2 receptor alpha chain on the surface of murine B cell-line B300-19. BALB/c mice were immunized with cells expressing the chimeric protein. The IgG purified from the resulting immune serum immunoprecipitated human c-Mpl. The immune IgG supported proliferation of both stable transfectant Ba/F3 cells expressing whole c-Mpl molecules (c-Mpl-Ba/F3 No. 9) and UT7/TPO cells bearing naturally occurring c-Mpl, whereas it did not support the growth of the untransfected parental Ba/F3 cells. Cell growth was induced using 3 to 100 microg/ml of immune IgG in a dose-dependent manner, but this induction was decreased at doses higher than 100 microg/ml. Non-immune IgG did not affect cell growth of c-Mpl-Ba/F3 No. 9 cells. Although the Fab fragment of immune IgG also immunoprecipitated c-Mpl, it did not support cell growth at concentrations as high as 180 microg/ml, implying that the bivalent binding of receptors by antibodies is essential for cell proliferation. These results suggest that antibodies against human c-Mpl stimulate the proliferation and differentiation of megakaryocytes by their bivalent binding to receptors like TPO.

Animals↗

PubMed1997

3-(13-Hydroxytridecyl)-1-[13-(3-pyridyl)tridecyl]pyridinium chloride (YM-53792), a novel inhibitor of NF-AT activation.

A compound, YM-53792, was identified as an inhibitor of interleukin-2 (IL-2) gene promoter activity, using a Jurkat cell-based reporter system in which the luciferase gene is regulated by the IL-2 gene promoter. Production of IL-2, interleukin-4 (IL-4) and interleukin-5 (IL-5) from human peripheral blood mononuclear cells was suppressed by YM-53792 in a dose-dependent fashion. Since expression of these cytokine genes is known to be regulated by NF-AT, we examined whether the promoter activity created by multimerization of NF-AT elements was inhibited with YM-53792. YM-53792 inhibited this promoter activity, but not AP-1- and NF-kappaB-driven promoter activities nor SV40 enhancer/promoter activity. In addition, electrophoretic mobility shift assays did not detect NF-AT/DNA complexes when nuclear extract prepared from YM-53792-treated, PMA/A23187-stimulated Jurkat cells was used, whereas AP-1/DNA complexes were observed. These results suggest that YM-53792 specifically inhibits the activation of NF-AT.

Cyclosporine↗

PubMed1997

Pro-apoptotic effect of the c-Abl tyrosine kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine.

Treatment of cells with the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) and other genotoxic agents is associated with activation of the c-Abl protein tyrosine kinase. The functional role of c-Abl in the response to DNA damage, however, remains unclear. The present studies demonstrate that cells expressing a dominant negative, kinase-inactive c-Abl (K-R) are resistant to killing by ara-C. The expression of c-Abl (K-R) blocked ara-C-induced apoptosis by a mechanism that is at least in part independent of the p53 tumor suppressor. Cells null for c-Abl also exhibited resistance to induction of apoptosis. These findings provide support for a pro-apoptotic function of c-Abl in the response to certain genotoxic drugs.

Apoptosis↗

PubMed1997

Inhibition of phosphatidylinositol 3-kinase by c-Abl in the genotoxic stress response.

Activation of phosphatidylinositol (PI) 3-kinase by growth factors results in phosphorylation of phosphatidylinositol lipids at the D3 position. Although PI 3-kinase is essential to cell survival, little is known about mechanisms that negatively regulate this activity. Here we show that the c-Abl tyrosine kinase interacts directly with the p85 subunit of PI 3-kinase. Activation of c-Abl by ionizing radiation exposure is associated with c-Abl-dependent phosphorylation of PI 3-kinase. We also show that phosphorylation of p85 by c-Abl inhibits PI 3-kinase activity in vitro and in irradiated cells. These findings indicate that c-Abl negatively regulates PI 3-kinase in the stress response to DNA damage.

Humans↗

PubMed1997

Role for E2F in DNA damage-induced entry of cells into S phase.

Mammalian cells respond to ionizing radiation (IR) with transient cell cycle arrest and induction of apoptosis. Here we show that IR increases the expression of the E2F-1 transcription factor and the entry of cells into S phase. E2F-1 transactivation function is inhibited by cyclin A-kinase to ensure orderly progression through S phase. However, in contrast to proliferating cells, IR treatment results in down-regulation of cyclin A-kinase. Expression of a dominant negative form of the E2F heterodimeric partner DP-1 confirmed the involvement of E2F in IR-induced S-phase entry. These findings also support opposing signals involving the induction of E2F and the down-regulation of cyclin A-kinase in the IR response.

Apoptosis↗

PubMed1997

Specific inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by liposomally endocapsulated antisense phosphorothioate oligonucleotides: penetration and localization of oligonucleotides in clone 76 cells.

Liposomally encapsulated phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated phosphorothioate oligonucleotides (S-ODNs) complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. Displacement of the target AUG initiation codon sequence to the 3'-end, 5'-end, and/or center sites on the antisense phosphorothioate oligonucleotides was studied with regard to the inhibition of influenza virus RNA polymerases and NP. The antisense phosphorothioate oligonucleotide containing the AUG initiation codon at the center site of the oligonucleotide had the highest inhibitory effects. The liposomally encapsulated phosphorothioate oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. Observation of clone 76 cells treated with the endocapsulated antisense phosphodiester oligonucleotide, FITC-ODNs-PB2-T3, by a confocal laser scanning microscope, revealed diffuse fluorescence, apparently within the cytoplasm. Interestingly, the endocapsulated antisense phosphorothioate oligonucleotide, FITC-S-ODNs-PB2-T3 accumulated in the nuclear region of clone 76 cells. However, weak fluorescence was observed in the endosomes and in the cytoplasms of the clone 76 cells treated with the free antisense phosphorothioate oligonucleotides.

Animals↗

PubMed1996

Inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by unmodified, phosphorothioated, and liposomally encapsulated oligonucleotides.

We have demonstrated that antisense phosphodiester (ODNs) and phosphorothioate oligonucleotides (S-ODNs) inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line, which is a derivative of the murine C127 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to treatment with dexamethasone. Phosphodiester, phosphorothioate, and liposomally encapsulated oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The ODNs and S-ODNs complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. On the other hand, the inhibitory effect of the S-ODNs targeted to PB1 was considerably decreased in comparison with the other three target sites. Liposome encapsulation afforded oligomer protection in serum-containing medium and substantially improved cellular accumulation. The liposomal encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. The activities of the unmodified oligonucleotides are effectively enhanced by using the liposomal carrier.

Animals↗

PubMed1996

Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyze RNA synthesis.

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. Enzyme reconstitution experiments indicated that all three P proteins are required for RNA synthesis in vitro (Kobayashi, et al. Virus Res 22, 235-245, 1992). Nuclear extracts of HeLa cells infected with three kinds of the recombinant vaccinia virus, each carrying one of the three P protein cDNAs, exhibited the activity of complete replication, that is, vRNA-sense RNA-directed and cRNA-sense RNA-directed RNA synthesis in the absence of primers. The nuclear extract from cells singly infected with the virus carrying PB1 cDNA exhibited a significant level of model v-sense RNA-directed RNA synthesis activity. Thus we conclude that PB1 is the catalytic subunit of influenza virus RNA polymerase and that under certain conditions, PB1 alone is able to catalyze RNA synthesis in vitro.

Base Sequence↗

PubMed1996

Immunoassay and immunohistology studies of chromogranin A as a neuroendocrine marker in patients with carcinoma of the prostate.

OBJECTIVES: Neuroendocrine differentiation in carcinoma of the prostate is characterized by the expression of neuroendocrine cell products such as chromogranin A (CgA). We studied serum levels and tissue staining for CgA in prostate cancer to assess their clinical value. METHODS: In 82 patients with prostate cancer, serum specimens were obtained at diagnosis and studied by both CgA and prostate-specific antigen (PSA) immunoassays. In 43 additional patients with prostate cancer, paraffin-embedded tissue from core biopsies or transurethral resections and serum samples were studied, respectively, by immunohistology and immunoassay for CgA. RESULTS: In serum samples from the 82 patients in whom CgA and PSA levels were measured, 26 of 82 (32%) had an elevated CgA (greater than 200 ng/mL), and 36 of 82 (44%) had an elevated PSA (greater than 4.0 ng/mL). Of the patients with Stage D2 cancer, 11 of 18 (61%) had an elevated CgA and 6 of 18 (33%) had an elevated PSA. Four of 5 patients with local recurrence had an elevated CgA, but only 1 patient had an elevated PSA. Of the 43 patients in whom serum and tissue CgA studies were performed, 12 (28%) had elevated serum CgA, and 15 of the 43 (35%) had CgA staining in their prostate tissue. Of the 14 of these patients with D2 disease (distant metastases), 9 (64%) had elevated serum levels of CgA and 6 (43%) had positive staining in their prostate tissue. Of the 9 patients with Stage D2 disease and elevated serum CgA, 6 had a normal serum PSA. CONCLUSIONS: Our studies complement those of others and indicate that CgA has potential as a clinically useful serum and tumor marker for prostate cancer. Serum CgA measurements can identify some patients with advanced disease who do not have elevated serum PSA. However, further studies in larger groups of patients are needed to define the clinical value of CgA as a marker for prostate cancer.

Biomarkers, Tumor↗

PubMed1996

Laparoscopic complications in markedly obese urologic patients (a multi-institutional review)

OBJECTIVES: Significant obesity is considered to be a relative contraindication to laparoscopic surgery. This study reviews the complications encountered in massively obese patients undergoing urologic laparoscopic surgery. METHODS: Body mass index (BMI) was used as an objective index to indicate massive obesity. Eleven institutions compiled retrospective data on 125 patients having a BMI greater than 30. Procedures performed included 76 pelvic lymph node dissections, 14 nephrectomies, 7 bladder neck suspensions, and 28 miscellaneous procedures. RESULTS: For the group as a whole, the mean BMI was 35.1 (range 30.1 to 57.2). Mean operative time was 202 minutes (range 60 to 480). Conversion to open surgery occurred in 15 of the 125 patients (12%). Complication rates (minor and major) were 22% (27 occurrences in 125 patients) intraoperatively and 26% (33 occurrences in 125 patients) postoperatively. The major complications included 2 trocar injuries to abdominal wall vessels, 1 bladder injury, 3 peripheral nerve injuries, 1 dysrhythmia, 1 deep vein thrombosis, 1 wound seroma, 1 nephrocutaneous fistula, 1 incisional hernia, and 1 death. CONCLUSIONS: In this review, complication rates for urologic laparoscopic surgery on massively obese patients were higher than in the general population undergoing laparoscopic surgery (0.3% to 21%).

Adolescent↗

PubMed1996

Herbimycin A down-regulates messages of cyclin D1 and c-myc during erythroid differentiation of K562 cells.

The ansamycin antibiotic, herbimycin A, is a potent tyrosine kinase inhibitor, and induces the erythroid differentiation of bcr-abl-possessing K562 cells. The growth of K562 cells was cytostatically reduced to less than 50% of the control level at 48 h by 0.5 microgram/ml of herbimycin A treatment. A total of 12% and 53% of the treated cells were benzidine-positive at 24 h and 48 h, respectively. The percentage of cells in the S phase decreased rapidly from 60% to 15% after 12 h of treatment. The reduction of S phase cells persisted until 24 h, whereas the G1 population conversely increased. Then underphosphorylated retinoblastoma gene product increased from 6 h to 24 h, but returned to baseline at 48 h. Most cell cycle controlling genes were unchanged by herbimycin A treatment. However, both cyclin D1 and c-myc were prominently down-regulated in the early phase of treatment, corresponding to the decline of the S phase population. Cyclin D1 was initially down-regulated to an undetectable level at 6 h, although its expression recovered gradually from 12 h and returned to baseline at 24 h. c-myc was also down-regulated from 1 h to 6 h. These data suggest that signals originating from bcr-abl kinase are at least partly transduced through both c-myc and cyclin D1, and that herbimycin A-induced erythroid differentiation occurs during or after the cessation of growth due to interference with these signals.

Antibiotics, Antineoplastic↗

PubMed1996

The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit is required for viral RNA synthesis in replication of the influenza virus genome.

We indicated that the PB1 and PA subunits of RNA polymerase and nucleoprotein (NP) can support replication of the influenza virus genome as well as transcription to yield uncapped poly(A)(+)-RNA (Y. Nakagawa, N. Kimura, T. Toyoda, K. Mizumoto, A. Ishihama, K. Oda, and S. Nakada, J. Virol. 69:728-733, 1995). To analyze the functions of the PB1 and PA subunits in replication and transcription, YP1N clones in which the PB1 and NP genes can be expressed in response to dexamethasone were established. cRNA was transcribed from model viral RNA (vRNA), but vRNA synthesis from model cRNA was not detected in YP1N clones. Furthermore, poly(A)(+)-RNA directed from model vRNA was synthesized in YP1N clones. These results indicated that PB1 and NP can support the syntheses of cRNA and poly(A)(+)-RNA and that the PA subunit, in addition to that of PB1 and to NP, is required for vRNA synthesis. In summary, the PB1 subunit is involved in the catalytic activities of nucleotide elongation, and the PA subunit may act as an allosteric modulator and cause a conformational change from a cRNA-to a vRNA-synthesizing form of the PB1 subunit.

Animals↗

PubMed1996

Quality of life and anxiety before and after lung cancer chemotherapy: relationship to patient's personality.

The purpose of the study was to assess the quality of life (QOL) and anxiety in 50 inpatients with primary lung cancer and examine the influence of their personalities on the QOL assessment. We used a psychological personality test to evaluate the patient's personality, then followed the course of QOL and anxiety before and after chemotherapy. To measure QOL, we used Holmes's QOL checklist, and the State-Trait Anxiety Inventory was adopted to estimate the patient's anxiety. Eighty courses of chemotherapy were administered, and QOL evaluations were performed 235 times. By using factor analysis, the somatic, social and psychological factors were extracted which confirmed the reliability and validity of the QOL checklist. The psychological QOL score showed a correlation with A (Adult) and AC (Adapted Child) of the five ego states in the Egogram. Therefore, it is important to survey the patients' personalities in order to grasp their QOL accurately.

Adult↗

PubMed1995

Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells.

Interferon (IFN) is one of the potent antiproliferative cytokines and is used to treat some selected cancers. IFN arrests the growth of Burkitt Lymphoma derived cell line Daudi cells in the G1 phase. G1-to-S progression is controlled by positive and negative regulatory genes. Therefore, we investigated the effects of IFN on G1-controlling genes. Expression of cyclin-dependent kinases (Cdks 2, 3, 4, 5, 6), MO15/Cdk7, and cyclins E and H was studied to assess positive regulators, while p15Ink4B, p16Ink4, p18, p21Cip1, and p27Kip1 were assessed as negative regulators. Cdks 2, 4, 6 and cyclin E were markedly down-regulated. MO15/Cdk7 expression showed little change, but its regulatory subunit (cyclin H) was down-regulated like cyclin E. Expression of p15Ink4B and p16Ink4 was not observed. p18 was induced until 48 h and its expression returned to the initial level at 72 h. In contrast, p21Cip1 mRNA expression remained at the baseline level throughout IFN treatment, while the expression of p27Kip1 increased at 48 and 72 h. Taken together, these data indicate that IFN changes the messenger RNA of G1-controlling genes towards the suppression of G1-to-S transition.

Base Sequence↗

PubMed1995

A macrolide antibiotic, roxithromycin, inhibits the growth of human myeloid leukemia HL60 cells by producing multinucleate cells.

The antiproliferative effect of roxithromycin (RXM) was studied using human myeloid leukemia HL60 cells. RXM inhibited the growth of HL60 cells in a concentration-dependent manner, and significantly inhibited growth at concentrations above 75 microM. This growth inhibition was not associated with specific cell cycle arrest and DNA synthesis was not impaired. In addition, the number of viable cells remained almost unchanged in the presence of 100 microM RXM. RXM induced growth inhibition at least partly by the formation of multinucleate cells. Both flowcytometric and morphological examination revealed that more than 40% of the RXM-treated cells were binucleate. These findings demonstrate that RXM is a potent new modulator of cell cycle progression in HL60 cells and suggest that the inhibition of cytokinesis by this drug may provide a new model for studying mitosis.

Bromodeoxyuridine↗

PubMed1995

The RNA polymerase PB2 subunit is not required for replication of the influenza virus genome but is involved in capped mRNA synthesis.

An established cell line, clone 64, in which the expression of the RNA polymerase PB1 and PA subunit genes and the nucleoprotein (NP) gene but not the PB2 subunit gene of influenza virus can be induced by the addition of dexamethasone, was used to analyze the replication and transcription machineries of the influenza virus. Both NS-CATc and NS-CATv, the chimeric nonstructural protein chloramphenicol acetyltransferase (NS-CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8 (the NS gene), respectively, can be transcribed into the corresponding complementary-strand RNA in clone 64 cells only when treated with dexamethasone. Although sense-strand poly(A)+ CAT RNA was detected in the dexamethasone-treated clone 64 cells transfected with NS-CATv RNA, CAT activity was not detected in these cells and the isolated poly(A)+ CAT RNA was inert in an in vitro translation system. However, when the poly(A)+ CAT RNA was capped by using a purified yeast mRNA capping enzyme (mRNA guanylyltransferase), the capped poly(A)+ CAT RNA became translatable in the in vitro translation system. These results indicated that PB1, PA, and NP can support the replication of the influenza virus genome as well as the transcription to yield uncapped poly(A)+ RNA and that PB2 is specifically required for the synthesis of capped RNA.

Animals↗