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S Nakada

Publications and source records attributed to S Nakada.

At least 19 recordsLinked to original sources
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

Transcription of a recombinant influenza virus RNA in cells that can express the influenza virus RNA polymerase and nucleoprotein genes.

A new transfection system for influenza virus was developed using the clone 76 cell line, in which the viral RNA polymerase and nucleoprotein (NP) genes can be expressed in response to dexamethasone. Ribonucleoprotein (RNP) complexes were reconstituted by expressing proteins from a chimeric NS-chloramphenicol acetyltransferase (CAT) RNA consisting of the full-length negative-strand RNA of the CAT gene positioned between the 5'- and 3'-terminal sequences of influenza virus RNA segment 8, and purifying NP from an NP gene-expressing Escherichia coli strain. When the reconstituted RNP was transfected into clone 76 cells, CAT was produced only when the synthesis of the three RNA polymerase subunits and NP was induced by treatment with dexamethasone.

Animals↗
PubMed1992

Induction of E1A-responsive negative factors for transcription of the fibronectin gene in adenovirus E1-transformed rat cells.

The level of fibronectin (FN) gene expression is very high in resting rat 3Y1 cells but greatly decreased in adenovirus E1-transformed cells. To study the mechanism of this down-regulation, nuclear factors binding to the 5'-flanking region of the FN gene were analyzed by gel retardation assay and DNase I footprinting. Nuclear factors that were present in the transformed cells but nearly absent in resting 3Y1 cells interacted with multiple sites of the promoter region. Oligonucleotide competition with the FN promoter-chloramphenicol acetyltransferase (CAT) reporter constructs (pFCAT) for these factors in the transformed cells indicated that all of them had a negative effect on FN gene expression. Of them, a factor(s) (G10BP) binding to the G10 stretch from positions -239 to -230 and to two GC boxes consisting of the G10 stretch with one internal C residue insertion from positions -105 to -95 and -54 to -44 had the strongest repressive activity. Introduction of substitutive mutations into these G-rich sequences resulted in the increase in CAT activity of pFCAT in the transformed cells. The recognition sequences of G10BP and Sp1 overlap in two GC boxes. G10BP has stronger affinity for heparin and GC boxes than does Sp1, suggesting that G10BP may repress FN gene transcription by displacing Sp1.

Adenoviridae↗
PubMed1992

E1A-responsive elements for repression of rat fibronectin gene transcription.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression. To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene. Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Sp1 motif GGGCGG. Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter. Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression. These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A.

Adenovirus Early Proteins↗
PubMed1992

A new model for evaluating corneal wound strength in the rabbit.

Presented in this report is a new model to evaluate corneal wound strength quantitatively by using an Instron 1125 tensiometer with video monitoring. Twenty-six rabbits received full thickness 8 mm central corneal incisions that were marked with sutures at each end and dressed with collagen shields. Animals were killed and corneas were harvested from 3-30 d postoperatively. Tensile test specimens, of rectangular (n = 9) or hourglass (n = 17) planform geometry, were prepared. Uniaxial tension tests were performed on these specimens parallel to the long dimension and perpendicular to the incision. Specimens were processed for light microscopy. Individual specimen load and displacement records were normalized to yield geometry independent stress and strain data sets. Maximum wound strength was determined as peak tissue stress on loading. Maximum tissue stiffness (inverse compliance) was derived from the peak slope of the stress-strain profile. Tissue fracture toughness was calculated as the total area under the stress-strain profile from the onset of loading to total wound failure. Data points at 3, 5, 10, 18, and 30 d show maximum wound strength increasing from 15.2 to 832 kilopascals (KPa), maximum stiffness increasing from 0.20 to 11.4 megapascals, and fracture toughness increasing from 1.82 to 87.7 KPa. Histological observations correlate well with tensiometry deductions. These data suggest enhanced mechanical load bearing with time, indicative of enhanced collagen fiber formation and cross-linking.

Animals↗
PubMed1991

Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1.

Normal human diploid fibroblasts, TIG-1, which have a replicative life span of about 62 population doublings (PD), tended to senesce after about 50 PD with a gradual decrease in sensitivity to serum. Treatment of TIG-1 cells with the antisense-Rb oligomer, which completely depleted the retinoblastoma susceptibility gene product (RB), extended life span by about 10 PD. Treatment with the antisense-p53 oligomer alone had no effect; however, cotreatment with the antisense-Rb oligomer further potentiated the extension and the increased sensitivity to serum caused by the antisense-Rb oligomer alone, suggesting that p53 and RB function in separate, yet complementary pathways in signal transduction to senescence. The c-fos expression, which is presumed to be regulated negatively by RB, was not stimulated in partially senescent TIG-1 cells by treatment with the antisense-Rb oligomer.

Base Sequence↗
PubMed1991

Interaction of nuclear factors with the regulatory region of the N-myc gene during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma cell line, NEC14, can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). During the early stage of HMBA-induced differentiation, the level of N-myc expression decreased steeply and transiently, and then quickly returned to its original level after reaching a minimal level at 18 h after addition of HMBA. Nuclear run-on experiments indicated that this transient decrease is regulated at the transcription start point. To investigate the mechanism of this down-regulation, the 5'-flanking region of the human N-myc gene was cloned and sequenced. Computer analysis of the sequence revealed high homology with the 5'-flanking region of the mouse N-myc gene, especially (greater than 80%) in the region of nt positions -1777 to -1732, nt positions -763 to -501 and nt positions -260 to + 1. The patterns of protein binding to the upstream region during the early stage of NEC14 cell differentiation were analyzed by gel retardation assay. The DNA fragments VIII and X, containing the sequences of nt positions -1437 to -1237 and nt positions -1863 to -1710, respectively, formed the DNA-protein complexes which were greatly reduced in quantity in the cell extract prepared 18 h after the addition of HMBA. This reduction, however, was not observed with an extract similarly prepared from the NEC14 derivative cell line, H10, expressing the N-myc gene constitutively. These results suggest a causal connection between the complex formation and the high-level transcription of the N-myc gene.

Acetamides↗
PubMed1991

Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to Latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrifugation was hybridized repeatedly to the cDNA-oligo(dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned into pBluescript II KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA inserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.

Acetamides↗
PubMed1991

Growth complementation of influenza virus temperature-sensitive mutants in mouse cells which express the RNA polymerase and nucleoprotein genes.

In order to establish cell lines which complement the growth of temperature-sensitive (ts) mutants of influenza virus, three RNA polymerase and nucleoprotein (NP) genes each linked to the mouse mammary tumor virus LTR were cloned into the bovine papillomavirus vector DNA. After co-transfection of mouse C127 cells with these recombinant plasmids, a cell line, clone 76, in which the expression of the three polymerase and NP genes could be stimulated by dexamethasone, was established. The clone 76 cells could complement the growth of ts-mutants defective in one of the polymerase subunit genes at the nonpermissive temperature in response to dexamethasone. The results suggest that the simultaneous expression of the three polymerase genes in the same compartment of protein synthesis machinery is required for an efficient complementation of ts-mutant growth.

Animals↗
PubMed1991

A transient decrease in N-myc expression and its biological role during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line, NEC14 can be induced to morphologically differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA) in vitro. The expression of several cellular oncogenes (c-onc) in NEC14 cells was examined after induction of differentiation by HMBA. The level of N-myc expression was the highest in undifferentiated cells but decreased transiently to less than 1/10 of the original level shortly after the induction of differentiation. To investigate the role of the transient decrease in N-myc level on NEC14 cell differentiation, a chimeric human N-myc gene in which transcription is initiated at the human beta-actin gene promoter was constructed and introduced into NEC14 cells. Several transformants expressing the exogenous N-myc gene constitutively were established. These transformants showed 10- to 70-fold increases in plating efficiency and shorter population doubling times as compared with the parental NEC14 cells. The transformants were hard to induce, spontaneously differentiated cells on the periphery of cell clusters in culture, unlike parental NEC14 cells, and took longer for HMBA-induced morphological differentiation. The populations of the cells expressing HLA and SSEA-1 antigens increased from 10%-20% to nearly 100% in NEC14 cells after the induction of differentiation, while the populations expressing these antigens increased only to 50%-60% in one of the transformants, S11. The transformants gained an increased tumorigenic potential in nude mice, and the tumors produced consisted exclusively of EC stem cells. These results suggest that the additional expression of the exogenous N-myc gene (increased about two-fold) confers the more transformed state on the cells.

Antigens, Surface↗
PubMed1990

A rapid and efficient cloning method with a solid-phase DNA probe: application for cloning the 5'-flanking region of the gene encoding human fibronectin.

A rapid and efficient gene-cloning method was developed using oligodeoxyribonucleotide probes (approx. 30-mer) immobilized by their 5' terminal to the gel (LiChrosphare Si1000; used for high-performance liquid chromatography). The method allowed us to concentrate DNA fragments having a particular base sequence in an eppendorf tube with very high efficiency within 2 h. For example, genomic DNA encoding the human fibronectin gene (fn) was concentrated by this method and cloned into lambda Charon4A vector. Screening of the concentrated genomic DNA library for the 5' region of the human fn gene showed that approx. 30% of 3000 recombinant phages tested were positive clones. Various factors affecting cloning efficiency were investigated.

Base Sequence↗
PubMed1990

Two-step transformation of rat 3Y1 cells by the adenovirus E1A and E1B genes.

The transformation of rodent cells by the adenovirus E1A and E1B genes was very efficient when these genes were physically linked. When they were cleaved, the transformation became very inefficient. To clarify this difference, the chimeric E1B genes in which either the adenovirus enhancer or the human beta-actin promoter was linked to the 5' side of the E1B gene were introduced into rat 3Y1 cells. The saturation density of these cell lines (eB or APrB) was similar to that of parental 3Y1 cells. When eB or APrB cell lines were supertransfected with the E1A gene, discrete dense foci were developed after 5-6 weeks, while the supertransfection of 3Y1 derivative cell lines, in which the enhancer-unlinked E1B gene was introduced, did not develop any dense foci. Analysis of the E1A and E1B transcripts in these cell lines indicated that the E1B gene is efficiently expressed in the presence of the E1A gene products if the enhancer is linked to the E1B gene and that an increased level of E1B proteins is required for an efficient expression of the E1A gene. These results indicated that E1A and E1B genes in separate pieces of DNA are capable of cooperatively transforming 3Y1 cells if appropriate cis-acting elements are attached and high-level expressions are achieved.

Actins↗
PubMed1990

Expression of various viral and cellular enhancer-promoters during differentiation of human embryonal carcinoma cells.

Alterations in the pattern of gene expression were studied during differentiation of the human embryonal carcinoma (EC) cell line NEC14. NEC14 cells can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The efficiency of DNA transfection of undifferentiated and differentiated NEC14 cells was compared by measuring the activities of endogenous and exogenously introduced promoters for the beta-actin gene and heat shock protein 70 gene. The results indicated that the efficiency was not significantly different in cells of these two states. Under the conditions used, all the viral enhancer-promoters tested showed very little or no activity in undifferentiated cells, but activities of SV40, BKV, adenovirus and RSV enhancers were greatly increased after differentiation. Activities of these viral enhancers in differentiated cells were completely repressed by cotransfection with the adenovirus E1A gene. An E1A-inducible promoter of the adenovirus E2 gene showed stronger activity in differentiated than in undifferentiated cells, and was not activated efficiently by cotransfection with the E1A gene in either undifferentiated or differentiated cells. These results indicate that factor(s) regulating activities of various enhancer-promoters in NEC14 cells is or are different from E1A-like factor(s) present in mouse EC F9 cells.

Acetamides↗
PubMed1990

Constraction of a subtracted cDNA library using oligo(dT)-latex.

An efficient method for constraction of subtracted cDNA library was developed using oligo(dT 30. Latex and PCR. This method improved the chances for identifying cDNA clones corresponding to scarce class of mRNA that is expressed differentially during cellular growth and differentiation.

Acetamides↗
PubMed1989

[Measurement of IgE, IgG1 and IgG4 antibodies against mite Sephadex fractions and purified allergens by means of enzyme-linked immunosorbent assay].

Mite antigens (Dermatophagoides farinae) were fractionated by a Sephadex G-200 column and their reactivities with IgE, IgG1 and IgG4 antibodies were investigated with enzyme-linked immunosorbent assay (ELISA). High IgE antibody values were observed in fractions with low molecular weight (allergenic part), while high IgG1 and IgG4 antibody values were observed in fractions with high molecular weight. High IgG4 antibody values to crude mite extract and fractions with high molecular weight were detected in individuals who had received immunotherapy. However, IgG4 antibodies directed to allergenic part were found in only one out of 12 sera tested. IgG4-ELISA using DF1 (major allergen of Dermatophagoides farinae) as antigen was also performed. In the group treated with mite, significant IgG4 antibody levels were detected in only one out of 13 sera tested. In the group treated with house dust, significant IgG4 antibodies were detected in only one out of 12 sera tested. Patients who showed high IgG4 antibody responses to crude mite extract and to high molecular weight did not show responses to allergenic part and DF1. The only case who showed positive IgG4 responses to allergenic part also reacted with DF1. Those results suggest that IgG1 and IgG4 antibody values in ELISA using crude mite extract as antigen do not reflect major allergen-specific antibody values. The importance of the use of partially purified antigens in measuring major allergen-specific IgG4 antibodies was also suggested.

Allergens↗
PubMed1988

Molecular cloning and characterization of cellular genes whose expression is repressed by the adenovirus E1a gene products and growth factors in quiescent rat cells.

Several cDNA clones of cellular genes, whose expression is repressed by the adenovirus type-12 E1a gene products, were isolated from a rat 3Y1 cell cDNA library by differential plaque hybridization with labeled cDNA probes prepared from 3Y1 and the derivative cell line expressing the E1a gene constitutively. The changes in the levels of these gene transcripts during cell-cycle progression from G0 to G1 to S phase were analyzed with 3Y1 cells and gMA cell lines, derived from 3Y1 cells, in which the expression of the E1a gene or its 13S, 12S cDNA can be switched on by the addition of dexamethasone. Quantitation of the transcripts by Northern-blot hybridizations and by nuclear run-on experiments revealed the following. (i) The level of clone-53 mRNA (which turned out to be the fibronectin (FN)-coding mRNA) is very high in resting gMA cells and decreased rapidly after switching on of the E1a gene or its 13S, or 12S cDNA. (ii) The addition of serum or platelet-derived growth factor to resting 3Y1 cells also resulted in a rapid decrease in the FN mRNA level, but the addition of epidermal growth factor (EGF) had little or no effect. (iii) The level of clone-56 mRNA in gMA cells was not affected by the induction of the E1a gene expression; however, the addition of EGF to resting gMA or 3Y1 cells resulted in a decrease of this mRNA after a 12- to 16-h lag period. Induction of the E1a gene expression in gMA cells treated with EGF shortened the lag period. The addition of serum to resting 3Y1 cells decreased the clone-56 mRNA level without a significant lag period.

Adenoviridae↗