[Functions of influenza virus RNA polymerase subunits].
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Animals↗
Biomedical subjects
S Nakada
Publications and source records attributed to S Nakada.
[Transformation into chronic myelomonocytic leukemia in a patient with primary myelofibrosis associated with severe hypoplasia: report of an autopsy case].
A 70-year-old male was admitted because of anemia in September 1989, and primary myelofibrosis was diagnosed based on the presence of leukoerythroblastosis, a normal chromosomal analysis and pathological findings of fibrosis in bone marrow. Although he was anemic, he did not require any treatment for two years. Then his hematological status deteriorated to severe pancytopenia, and the marrow biopsy revealed marked hypoplasia with fatty replacement and scattered fibrosis. He was treated with metenolon without success and frequent transfusion of packed red cell was required. This hypoplastic status continued for seven months. In May 1992 his WBC count increased gradually with monocytosis. The marrow was filled with various stages of monocytes, with almost no fibrosis remaining. The chromosomal analysis was repeated but disclosed no abnormalities, consistent with the negative result of BCR-ABL rearrangement investigated by the RT-PCR method. One month later, when the patient died of multiple cerebral bleeding and infection, the leukocyte count exceed 90,000/microliters. It is known that major causes of death for patients with primary myelofibrosis are infection, bleeding, cardiac trouble and transformation to leukemia. We describe a case of myelofibrosis who developed to chronic myelomonocytic leukemia following severe aplastic phase.
Aged↗
Inhibition of influenza virus RNA polymerase and nucleoprotein of gene expression by antisense oligonucleotides.
We demonstrated that unmodified and modified (phosphorothioate) antisense oligonucleotides inhibit CAT (chloramphenicol acetyltransferase) protein expression in the clone 76 cell line. This cell line expresses the influenza virus RNA polymerase and nucleoprotein (NP) genes in response to dexamethasone. Antisense oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for the their inhibitory effects by a CAT-ELISA assay. Antisense phosphorothioate oligonucleotides (S-ODNs) complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed a high inhibitory effect. 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.
Base Sequence↗
Regulation of influenza virus RNA polymerase activity by cellular and viral factors.
An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.
Biological Factors↗
Changes of cell cycle-regulating genes in interferon-treated Daudi cells.
Interferon (IFN) modulates the expression of several genes and some of them are considered to be responsible for the inhibition of cellular growth. However, the alterations of cell cycle-regulating genes produced by IFN still remain unclear. Accordingly, we studied the expression of cell cycle-regulating genes during IFN-induced growth arrest. Cell cycle synchronized and unsynchronized Daudi Burkitt lymphoma cells were treated with IFN. Both the cell cycle distribution and the expression of cell cycle-regulating genes (cdk2, cdc2, cyclins A, B, C, D3, cdc25, and wee 1) were studied by flow cytometry and by Northern blot hybridization or the reverse-transcription polymerase chain reaction, respectively. Treated cells passed through the first G1 phase and gradually accumulated in the following G1 phase. Expression of cyclins A, B, and D3 oscillated along with the cell cycle progression in control cells, and the alterations of cyclin B expression were especially prominent. Both cdc2 and cdk2 also showed changes, but these were not so distinct as observed with cyclin B. Expression of cdc25 and wee1 was little affected by cell cycle progression. In IFN-treated cells, expression of cyclins A and B were down-regulated, while that of cyclin C was not. Cyclin D3 expression was also down-regulated at 48 h, followed by an increase at 72 h. Expression of both cdc2 and cdk2 was down-regulated, especially that of the later. Wee1 expression was down-regulated by IFN but, the expression of cdc25 remained stable. These findings suggest that the modulation of cell cycle-regulating genes, particular by cyclin A and cdk2, plays an important role in IFN-induced cellular growth arrest.
Base Sequence↗
Changes of G1 cyclins, cdk2, and cyclin A during the differentiation of HL60 cells induced by TPA.
Differentiation induction by 12-o-tetradecanoyl 13-acetate (TPA) results in the growth arrest of HL60 cells in the G1 phase. However, little is known about the changes of cell cycle-regulating genes during this differentiation process. We investigated the changes of mRNA for various cyclins (A, C, D1, D2, D3 and E) and cdk2. Synchronized HL60 cells began to proliferate immediately after release from cell cycle block and cell cycle synchrony was obvious until the second S phase. TPA-treated cells accumulated in G1 phase within 24 h and most of the cells were arrested in this phase at 36 h. The expression of cyclins and cdk2 was studied by Northern blot hybridization of the reverse-transcription polymerase chain reaction (RT-PCR). TPA treatment altered the expression of all genes studied. The expression of cdk2 and cyclin A mRNA was markedly down-regulated. Cyclin E mRNA expression was also prominently down-regulated from 12 h to 36 h, at which time a second increase of its expression was observed in control cells. In contrast, the expression of cyclin D1 mRNA was induced by TPA, while its expression in control cells was undetectable by Northern blot hybridization throughout the cell cycle. Cyclin C expression was faint and fluctuated irrelevant of cell cycle, but its expression in both control and TPA-treated cells was higher than at baseline. Cyclin D2 expression remained stable in control cells and TPA treatment resulted in slight down-regulation at 12 h, but no difference was observed after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)
Base Sequence↗
Influenza virus NS1 protein stimulates translation of the M1 protein.
The influenza virus NS1 protein was shown to stimulate translation of the M1 protein. M-CAT RNA, which contains the chloramphenicol acetyltransferase (CAT) reporter gene and the terminal noncoding sequence of segment 7 (coding for the M1 and M2 proteins), was ribonucleoprotein transfected into clone 76 cells expressing the influenza virus RNA polymerase and NP proteins required for the transcription and replication of influenza virus ribonucleoproteins. When the cells were superinfected with a recombinant vaccinia virus which expresses the NS1 protein, CAT expression from the M-CAT RNA was significantly stimulated but transcription was not altered. The expression of NS-CAT RNA, which contains noncoding sequences of segment 8 (coding for the NS1 and NS2 proteins), was not altered by the NS1 protein. Site-directed mutagenesis showed that the sequence GGUAGAUA upstream of the initiation codon on segment 7 was required for stimulation.
Base Sequence↗
[Noninvasive evaluation of chronic cumulative and acute cardiotoxicity induced by anthracycline in children with acute leukemia. Assessment of mainly left ventricular contractile state by M mode echocardiography].
The cardiotoxic effects of Daunomycin (DM) ad Adriamycin (ADR), Anthracyclines (ATC), were studied mainly by echocardiography to evaluate their chronic cumulative cardiotoxicity and acute cardiotoxicity during consolidation therapy. Electrocardiographic findings were less sensitive and therefore less reliable for evaluation of the chronic cumulative cardiotoxicity and acute cardiotoxicity induced by ATCs during consolidation therapy. With echocardiography ESS/ESVI, the index of the left ventricular contraction, had the highest sensitivity among indices for cardiotoxicity. SF was useful for easy measurement and calculation of cardiotoxicity. Evaluation of the chronic cumulative cardiotoxicity of ATC drugs indicated that cumulative doses of 300 mg/m2 or more resulted in abnormally low ESS/ESVI levels suggesting cardiotoxicity in many cases, and doses of 500 mg/m2 or more caused abnormally low levels in all cases. Evaluation of acute cardiotoxicity showed that post-consolidation therapy ESS/ESVI levels were significantly lower than pre-consolidation therapy levels in the group treated with doses of 500 mg/m2 or more, but the condition was reversible except in those patients with heart failure. As for the relation between cumulative doses and cardiotoxicity, the indices studied showed no differences between DM and ADR. These results indicate that careful follow-up mainly by echocardiography is required after doses reaching 300 mg/m2 or more of ATC drugs.
Adolescent↗
[Blood-transfusion in Vietnam].
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Blood Donors↗
Identification of a universal B cell epitope on DNA topoisomerase I, an autoantigen associated with scleroderma.
OBJECTIVE: To investigate the distribution of B cell autoepitopes of human DNA topoisomerase I (topo I), an autoantigen associated with scleroderma. METHODS: A complementary DNA clone, T1B, was used to produce recombinant proteins of topo I as beta-galactosidase fusion proteins. Immunoreactivity to these fusion proteins was then tested in 35 anti-topo I-positive sera from patients with scleroderma, by immunoblotting, enzyme-linked immunosorbent assay, and double immunodiffusion. RESULTS: One epitope was found to be universally recognized by all sera tested. Thirty-two of the samples recognized multiple antigenic regions, but sera from the remaining 3 patients recognized only this universal epitope, and in longitudinal studies of 1 of these 3 patients, the serum recognized only this epitope for more than 2 years, even though multiple, potent, antigenic regions were found on topo I. CONCLUSION: Recognition of multiple epitopes in most patients suggests that the topo I molecule itself would drive the autoimmunity on topo I. However, antigen-driven autoimmunity could not explain the production of the monoreactive anti-topo I antibody seen in the 3 patients. We thus hypothesize that there is a process whereby recognition of the universal epitope by cross-reaction develops into antigen-driven autoimmunity.
Autoantibodies↗
Differential activation of cyclin and cyclin-dependent kinase genes by adenovirus E1A12S cDNA product.
The differential activation of cyclin and cyclin-dependent kinase genes by the adenovirus E1A gene product (E1A) or serum factors was studied with a rat 3Y1 derivative cell line, g12-21, in which the E1A12S cDNA can be expressed in response to dexamethasone (dex). The induction of DNA synthesis in quiescent g12-21 cells occurred within 12 h after serum stimulation, while it occurred within 8 h after treatment with dex. The expression of cyclin D1 and E genes in the serum-stimulated cells was induced in mid G1 and mid to late G1, respectively, while that of the cyclin D1 gene was not induced and the induction of the cyclin E gene was shifted to the G1/S boundary in the dex-treated cells. The cdk2 gene was induced in late G1 and cdc2 and cyclin A genes at the G1/S boundary in both serum-stimulated and dex-treated cells. These results suggest that E1A skips cell cycle events which normally occur in early to mid G1 and may directly activate late-response genes. Analysis of the transcription factor E2F complexes formed in the promoter regions of cdc2 and dihydrofolate reductase genes showed that the amount of complexes formed is maximal at the G1/S boundary, but decreases in S phase when these genes are transcribed extensively.
Adenovirus E1A Proteins↗
Molecular assembly of influenza virus: association of the NS2 protein with virion matrix.
The NS2 protein, one of the two nonstructural proteins of influenza A virus, was found to exist in virus particles. By immunochemical method, the average number of NS2 in a virus particle was estimated to be 130-200 molecules. After solubilization of viral envelope, NS2 was still associated with ribonucleoprotein cores, but was later dissociated upon removal of the M1 protein. A filter-binding assay in vitro indicated direct protein-protein contact between M1 and NS2. Following chemical cleavage of the M1 protein, NS2 bound only a C-terminal fragment of M1. By immunoprecipitation, NS2-M1 complexes were also detected in virus-infected cell lysates. These observations altogether indicate specific molecular association between M1 and NS2. Biological significance of M1-NS2 interaction is discussed.
Animals↗
Tumour necrosis factor-alpha enhances cAMP-induced programmed cell death in mouse thymocytes.
During T-lymphocyte differentiation in the thymus, the majority of thymocytes die by apoptosis in situ. This process is characterized by internucleosomal DNA fragmentation and is induced by a number of stimuli including glucocorticoids, calcium ionophore, cAMP and 12-o-tetradecanoylphorbol 13-acetate (TPA). In this study, the effect of cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma) on the programmed cell death of thymocytes was examined by measuring DNA fragmentation and LDH release. TNF-alpha and IFN-gamma had no effect on DNA fragmentation in control and TPA, or A23187-treated thymocytes. Both human and murine rTNF-alpha enhanced cAMP-induced programmed cell death dose-dependently, but IFN-gamma had no effect on the process. TNF-alpha did not stimulate cAMP accumulation in control or 2-chloroadenosine-treated thymocytes. TPA markedly stimulated cAMP-induced DNA fragmentation as a result of 6 h incubation, whereas TNF-alpha did not. Thus TNF-alpha did not appear to activate protein kinase C directly. The effect of TNF-alpha was observed in the cell preparations from which adherent cells had been removed, suggesting that cytokines secreted by adherent cells in response to TNF-alpha are not involved in the process. The enhancement of cAMP-induced DNA fragmentation was observed in CD4+CD(8+)-double positive cells, but not in CD4+CD(8-)-single positive cells. The results of the present study indicate that a physiological cytokine, TNF-alpha, may modulate programmed cell death in immature thymocytes in concert with cAMP.
2-Chloroadenosine↗
An in vivo study of the replication origin in the influenza virus complementary RNA.
A new in vivo replication system for influenza virus was developed by using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein genes can be expressed in response to dexamethasone. The chimeric NS-chloramphenicol acetyltransferase (CAT) RNAs in the sense and antisense orientations positioned between the 5'- and 3'-terminal sequences of the influenza virus RNA segment 8 can be replicated [both genomic RNA (vRNA) and complementary RNA (cRNA) were transcribed] in the clone 76 cells treated with dexamethasone. These data indicate that three RNA polymerase proteins (PB1, PB2, and PA) and nucleoprotein are sufficient for replication of the influenza virus genome. Analysis of mutant cRNAs containing a base-substitution or a deletion in the 3'-conserved terminal 13 nucleotides revealed that important cis elements in the cRNA for vRNA synthesis reside at positions 2, 3, and 7 to 13 nucleotides from the 3'-end.
Animals↗
Protein phosphorylation required for the formation of E2F complexes regulates N-myc transcription during differentiation of human embryonal carcinoma cells.
The human embryonal carcinoma (EC) cell line NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The N-myc gene is expressed at a high level in the undifferentiated cells, but the level decreased steeply after 12-24 h HMBA treatment, returning to its original level after 48 h. The alteration in the N-myc level was well correlated with the formation of complexes with the E2F motif in the N-myc promoter region, and no complex was formed with cell extracts prepared from cells treated with HMBA for 12-24 h. The absence of E2F complexes during this period was caused by an inhibitor generated by a phosphatase reaction. Treatment of the 12-h extract with a cyclic AMP-dependent protein kinase resulted in the formation of E2F complexes, and treatment of the undifferentiated (0 h) and 48-h extracts with a calf intestinal phosphatase abolished complex formation completely. An inhibitor generated by the 0-h extract after treatment with a phosphatase inhibited E2F complex formation by the untreated 0-h extract in the presence of phosphatase inhibitors, okadaic acid and sodium vanadate. One of the two E2F complexes in the undifferentiated cells contained cyclin A, but the complex with similar mobility, formed after the transient decrease in the N-myc level, did not.
Carrier Proteins↗
Resonant transmission and velocity renormalization of third sound in one-dimensional random lattices.
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Journal Article↗
Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product.
The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.
Adenovirus Early Proteins↗
Hyperproduction of human interferon gamma by rat cells maintained in low-serum medium using the fibronectin gene promoter.
An expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter. Human interferon gamma (HuIFN-gamma) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established. These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence. The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4 x 10(5) IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days. The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks. HuIFN-gamma secreted into the medium had a molecular weight range of 22,000 to 25,000, similar to that of IFN-gamma produced by human lymphocytes. The N-linked glycosylation of HuIFN-gamma seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17,000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-gamma.
Animals↗