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A Tsuboi

Publications and source records attributed to A Tsuboi.

At least 73 records · Page 4Linked to original sources
PubMed1992

Effects of 5-fluorouracil on hematopoietic stem cells in normal and irradiated mice.

The effects of 5-fluorouracil (5-FU) on hematopoietic stem cells (CFU-S) and nucleated cells in mouse femur and spleen were studied in normal and X-irradiated mice (ddY-SLC male, 8-10 weeks old). Changes in the number of circulating blood cells also were investigated in mice treated with 5-FU. A single dose of 5-FU (150 mg/kg) was injected i.p. The femoral CFU-S decreased after 5-FU treatment up to day 3 when it reached 2% of the control value. The cells then increased, reaching a maximum about twice that of control by day 12. A very similar profile was found for splenic CFU-S. Post-irradiation recovery for femoral and splenic CFU-S in mice treated with 5-FU 3 days before X-irradiation (1.9 Gy) was faster than in mice given irradiation alone. Regrowth rates of CFU-S were almost the same as in mice treated with 5-FU alone. The radiosensitivity of the CFU-S population in mice treated first with 5-FU at different times before X-irradiation (1.9 Gy) changed day-by-day after treatment. The maximal survival for femoral CFU-S was found in mice treated with 5-FU 5 days before irradiation, and for splenic CFU-S in mice treated 12 days before irradiation.

Animals↗
PubMed1992

In vivo and in vitro interaction between interleukin 6 and granulocyte colony-stimulating factor in the regulation of murine hematopoiesis.

The interaction both in vitro and in vivo between human recombinant interleukin 6 (IL-6) and human recombinant granulocyte colony-stimulating factor (G-CSF) in the regulation of mouse hematopoiesis was investigated. In the in vitro experiments, mouse bone marrow and spleen cells were cultured in semisolid medium containing 5 or 50 ng/ml of G-CSF and concentrations ranging from 0 to 20 ng/ml of IL-6. In vivo, mice were treated for 4 days with 15, 50, or 250 micrograms/kg body weight/day of G-CSF, or with similar doses of G-CSF plus 50 micrograms/kg/day of IL-6, and the numbers of stem (spleen colony-forming units, CFU-S) and progenitor (megakaryocyte colony-forming cells, Meg-CFC; granulocyte-macrophage colony-forming cells, GM-CFC) hematopoietic cells and mature circulating blood cells were evaluated. In vitro IL-6 caused dose-dependent suppression of the proliferation of GM-CFC, decreasing numbers of granulocyte-macrophage colonies in culture. The inhibitory effect of IL-6 decreased along with the increase of density of cultured cells, suggesting the influence of accessory, cytokine-producing cells. In vivo, the numbers of GM-CFC and Meg-CFC in mice treated with IL-6 plus G-CSF were significantly closer to the values observed in untreated animals than those in mice treated with G-CSF only. The other cell populations were unaffected by IL-6 treatment. Our results demonstrate antagonism between IL-6 and G-CSF in the in vitro stimulation of the proliferation of late granulocyte precursors, and they suggest a similar effect in the in vivo regulation of granulopoiesis and megakaryocytopoiesis at the progenitor cell level.

Animals↗
PubMed1992

Transcriptional activator Tax of HTLV-1 binds to the NF-kappa B precursor p105.

Human T-cell leukemia virus type 1 (HTLV-1) induces adult T-cell leukemia and also a neurological disease, tropical spastic paraparesis. Tax protein (p40tax) of HTLV-1 activates in trans its own transcriptional enhancer in the long terminal repeat and also those in some cellular genes such as interleukin 2 receptor alpha, granulocyte-macrophage colony-stimulating factor, Fos, Jun and MHC class I. Thus, Tax has been proposed to play a critical role in the pathogenesis induced by HTLV-1 infection. Here, we report formation of a complex of Tax protein with the precursor protein p105 of the NF-kappa B p50 subunit. p105 was co-immunoprecipitated with Tax protein from cells infected with HTLV-1 from cells transfected with the Tax expression plasmid, but not from cells transfected with inactive mutants of Tax. Furthermore, a GST-p105 fusion protein produced in Escherichia coli bound to Tax protein. These results strongly suggest that the trans-activator Tax protein forms a complex with precursor NF-kappa B p105 and plays a role in trans-activation of transcriptional initiation.

Gene Products, tax↗
PubMed1992

In vivo administration of interleukin 6 delays hematopoietic regeneration in sublethally irradiated mice.

The in vivo effects of recombinant human interleukin 6 (IL-6) on the hematopoietic system of sublethally (4.8 Gy) x-irradiated mice were investigated. Animals were injected twice daily s.c. with IL-6 (10 micrograms/kg body weight/day) for 7 days following irradiation, and the numbers of hematopoietic stem, progenitor, and circulating blood cells were evaluated at 4, 7, 13, and 23 days. IL-6 caused significant depression of early hematopoiesis (decreased numbers of spleen colony-forming units [CFU-S] and granulocyte-macrophage colony-forming cells [GM-CFC]) in the spleens of irradiated mice. Marrow hematopoiesis was less affected by IL-6 injection, although the number of hematopoietic cells was also significantly lower than in irradiated mice injected with carrier alone. The observed decrease in the numbers of hematopoietic cells was not reflected by any significant change in the circulating blood cell numbers, which were similar to those in control irradiated animals. In contrast, IL-6 administered in 100 times higher doses (1000 micrograms/kg/day) caused significant increases in bone marrow and spleen cellularity and GM-CFC numbers, thus accelerating postirradiation hematopoietic regeneration. Our studies show that IL-6, administered in relatively low doses, suppresses postirradiation hematopoietic recovery, decreasing the numbers of stem and progenitor cells in sublethally irradiated mice.

Animals↗
PubMed1991

A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in responses to T-cell activation: purification from human T-cell leukemia line Jurkat cells and similarity to NF-kappa B.

Activation of T cells by antigen, lectin, or a combination of phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) leads to the induction of genes for a set of lymphokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF). We demonstrated in earlier studies that the upstream region of the mouse GM-CSF promoter at positions between -95 and -73 is essential for transcriptional activation in response to PMA/A23187. This region contains two DNA-binding motifs, GM2 and GC-box. The GM2 sequence (GGTAGTTCCC) is recognized by an inducible factor NF-GM2; the other (CCGCCC) by constitutive factors A1, A2, and B. To elucidate the mechanism of GM-CSF gene activation, we have purified the inducible factor NF-GM2 from the nuclear extract of stimulated Jurkat cells on the basis of specific DNA-binding activity. The purified NF-GM2 consists of 50 (p50) and 65 kDa (p65) polypeptides and has a binding activity specific for both the GM-CSF and immunoglobulin kappa (GGAAAGTCCC) enhancers. Electrophoretically purified p50 alone can form a protein-DNA complex, but in the mixture, p50 associates preferentially with p65 to form the NF-GM2 complex. In addition, p65 gave per se, with low affinity, a protein-DNA complex that migrated more slowly than native NF-GM2 complex. Furthermore, an antiserum against KBF1 (identical to 50 kDa NF-kappa B protein) reacted with the p50 of NF-GM2, indicating that the NF-GM2 polypeptide cannot be immunologically differentiated from the 50 kDa subunit of NF-kappa B. The purified NF-GM2 activated in vitro transcription from the kappa B enhancer, while it failed to stimulate transcription from the GM-CSF promoter harboring the GM2 sequence. This suggests that the activation mechanism of the GM-CSF gene through the GM2/GC-box sequence is different from that of genes carrying the kappa B enhancer alone.

Base Sequence↗
PubMed1990

In vivo effects of recombinant granulocyte-colony stimulating factor in athymic nude mice.

The possible role of T lymphocytes in in vivo regulation of haemopoiesis by recombinant human granulocyte-colony stimulating factor (rhG-CSF) was evaluated. Athymic nude (nu/nu) mice and their normal (+/+) littermates were injected subcutaneously twice daily with 100 micrograms/kg per day of rhG-CSF for 5 days. Such parameters as number of neutrophils in blood, spleen weight and cellularity, bone marrow cellularity, and number of stem and progenitor cells (colony forming units in spleen [CFU-S], mix colony forming cells [Mix-CFC], granulocyte-macrophage colony forming cells [GM-CFC] in bone marrow and spleen were evaluated. Some effects of the rhG-CSF treatment were similar in nu/nu and +/+ mice. Others, however, were to some extent different in the two groups of animals. It is concluded that T lymphocytes may be partially responsible for some of the effects of rhG-CSF in vivo activity.

Animals↗
PubMed1990

Inducible and non-inducible factors co-operatively activate the GM-CSF promoter by interacting with two adjacent DNA motifs.

The upstream region of the mouse granulocyte macrophage colony stimulating factor (GM-CSF) gene between positions -95 and -73 is required for phorbol-12-myristate-13-acetate (PMA)- and calcium ionophore (A23187)-inducible transcriptional activity in vivo. To study the mechanism of GM-CSF gene activation in T cells, nuclear extracts from non-stimulated and PMA/A23187-stimulated Jurkat cells were used in DNA binding assays. DNA mobility shift assays with wild type and mutant oligonucleotides revealed that this region contains at least two DNA binding motifs. One is the binding sequence GGTAGTTCCCC (positions -91 to -81), recognized by NF-GM2 (nuclear factor of GM-CSF 2), and the other is a GC-rich sequence (GC-box). NF-GM2, induced in Jurkat cells by PMA/A23187 stimulation, effectively competed with DNA containing the NF-kappa B binding sequence, suggesting that it has NF-kappa B-like activity. By UV cross-linking analysis, three cross-linked bands, corresponding to Mr 165, 70, and 60 kd for NF-GM2, and 110 and 130 kd for A1 and A2, respectively, were identified. Transfection experiments showed that activation of the GM-CSF gene in response to PMA/A23187 stimulation was abolished by point mutations in either the GC-box or the NF-GM2 site; these mutations also abolished binding of the respective proteins. These results indicate that constitutive (GC-box binding factor) and inducible (NF-GM2) factors regulate the GM-CSF promoter co-operatively in a PMA/A23187-inducible manner in vivo.

Animals↗
PubMed1990

Conserved structures of cell wall protein genes among protein-producing Bacillus brevis strains.

Bacillus brevis HPD31 contains a surface (S)-layer protein, termed the HWP, which forms a hexagonal array in the cell wall. The 5' region of the HWP gene was isolated from a DNA library constructed in bacteriophage vector EMBL3 from a partial BamHI digest of the chromosomal DNA. The 3' region contained in a 2.7-kilobase BglII fragment of the DNA was cloned into Escherichia coli, using pUC118 as a vector. On the basis of the chemically determined N-terminal amino acid sequence, the HWP gene was found to encode a polypeptide consisting of 1,087 amino acid residues with a signal peptide of 53 or 23 amino acid residues. The deduced amino acid composition was similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic relative to basic amino acids and in the very low content of sulfur-containing amino acids. The deduced amino acid sequence showed high homology (78%) with that of the middle wall protein of B. brevis 47. Furthermore, the multiple 5' ends of the HWP gene transcripts detected on S1 nuclease analysis closely resembled those of the middle wall protein gene transcripts. This complex structure was also conserved (greater than 85%) in the regulatory regions of two other cell wall protein genes isolated from B. brevis HPD52 and HP033, suggesting that the synthesis of the cell wall proteins is intricately regulated through a similar mechanism in protein-producing B. brevis.

Amino Acid Sequence↗
PubMed1990

Water content in cultured mammalian cells for dosimetry of beta-rays from tritiated water.

One of the critical factors for dosimetry of beta-rays from tritiated water is the water content within the cell. We estimated the cellular water concentration in cultured mammalian cells by measuring accurately the fraction of the extracellular water in the cell sample with [14C]inulin. The net water content (ml.g-1) after correcting for the extra-cellular water fraction was 0.858 for HeLa (human), 0.833 for JTC12P3 (monkey), 0.829 for NRK (rat), 0.843 for C3H10T1/2 (mouse) and 0.846 for L5178Y (mouse) cells. The mean water content (+/- S.E.M.) in these 5 lines of cultured mammalian cells was 0.842 +/- 0.005.

Animals↗
PubMed1990

In vivo effects of human recombinant interleukin 6 on hemopoietic stem and progenitor cells and circulating blood cells in normal mice.

Recombinant human interleukin 6 (rhIL-6) was administered s.c. every 12 h at a daily dose of 10 micrograms/kg body weight to normal healthy mice. After 4 days the numbers of progenitor cells (erythroid burst-forming units, BFU-E and granulocyte-macrophage colony-forming cells, GM-CFC) were significantly increased (p less than 0.01) in the bone marrows and spleens of treated animals. There was no significant change in spleen colony forming unit (CFU-S) number, whereas mixed-lineage colony-forming cell (Mix-CFC) number was elevated only in bone marrow. The number of nucleated cells in peripheral blood was increased in rhIL-6-treated mice, resulting from a significant (p less than 0.01) increase in neutrophil numbers and a decrease in lymphocyte numbers. The number of platelets in these animals was also higher than in controls (p less than 0.05). These results suggest that rhIL-6 is an effective stimulator of unipotent hemopoietic cells of myeloid, erythroid, and thrombocytic lineages when administered in vivo to mice and indicate a possible therapeutic potential of IL-6 in clinical situations.

Animals↗
PubMed1989

Isolation of a cDNA encoding Aspergillus oryzae Taka-amylase A: evidence for multiple related genes.

Complementary and genomic DNAs encoding Aspergillus oryzae Taka-amylase A (Taa) were cloned and sequenced. The coding sequence of the cDNA comprised the signal peptide [21 amino acids (aa)] and mature Taa (478 aa). The deduced aa sequence agrees well with the published aa sequence, except for one insertion, one deletion and ten aa substitutions. These differences might be due to the difference in the strains used. Sequence comparison of the cDNA and genomic DNA indicates the presence of eight introns ranging in size from 55 to 86 bp. Southern-blot analysis showed the presence of at least two Taa genes, and the second gene (Taa-G2) was isolated. All the intron/exon junctions follow the 'GT-AG' rule, except for intron I of the first gene (Taa-G1). The 5'-noncoding region was well conserved among the genomic genes and contained sequences similar to 'CAAT' and 'TATA' boxes at nucleotides -121 and -31, counted from the transcription start point, respectively. The 3'-noncoding regions, however, differed significantly from each other. Taa-G2 contains a sequence identical to that of several independent cDNA clones, suggesting that it may be the major transcribed gene in A. oryzae.

Amino Acid Sequence↗
PubMed1989

Three-dimensional structure of the surface protein layer (MW layer) of Bacillus brevis 47.

The three-dimensional (3D) structure of one surface protein layer from Bacillus brevis 47, the middle wall (MW) layer, has been reconstructed from tilted-view electron micrographs after correlation averaging to a resolution of 2 nm. The MW layer has p6 symmetry with a center-to-center spacing of 18.3 nm and a minimum thickness of 5.5 nm. The reconstruction reveals a distinct domain structure: the heavier domain of six monomers jointly forms a massive core centered at the sixfold symmetry axis, and lighter domains interconnect adjacent unit cells. In addition, the larger domains collectively form a pore by making contact with each other towards the inner surface, while the smaller domains establish a second connectivity towards the outer surface of the S layer. The MW layer of B. brevis resembles the S layer of Acetogenium kivui in various aspects: they have very similar lattice parameters and highly reminiscent 3D structures; the pores penetrate through the whole core and appear to determine the porosity of the S layers.

Bacillus↗
PubMed1989

In vitro reconstitution of a hexagonal array with a surface layer protein synthesized by Bacillus subtilis harboring the surface layer protein gene from Bacillus brevis 47.

Bacillus brevis 47 contains two surface layer proteins, termed the outer wall protein and the middle wall protein (MWP), which form a hexagonal array in the cell wall. Introduction of the MWP structural gene into Bacillus subtilis by using a low-copy-number plasmid led to the synthesis of an immunoreactive polypeptide with a molecular mass almost the same as that of the MWP synthesized by B. brevis 47. Biochemical analysis indicated that most of the MWP synthesized by B. subtilis was localized in the cytoplasmic fraction. This was further confirmed by using immunogold electron microscopy. The amino-terminal amino acid sequence of the MWP purified from the cytoplasm of B. subtilis indicated that the MWP was precursor with a signal peptide of 23 amino acid residues to the amino terminus of the mature protein. The precursor of the MWP possessed the ability to reassemble in vitro on the B. brevis 47 peptidoglycan layer, resulting in the formation of almost the same hexagonal arrays as with the mature MWP purified from B. brevis 47, judging from images averaged at a resolution of about 2.5 nm. Furthermore, a center-to-center distance of the hexagonal lattice on the envelope reconstituted by using the precursor MWP was calibrated as 18.3 nm, which was almost identical to the value of 17.8 nm obtained with the mature protein.

Amino Acid Sequence↗
PubMed1988

Characterization of the genes for the hexagonally arranged surface layer proteins in protein-producing Bacillus brevis 47: complete nucleotide sequence of the middle wall protein gene.

Bacillus brevis 47 contains two surface (S)-layer proteins, termed the outer wall protein (OWP) and the middle wall protein (MWP), which form a hexagonal array in the cell wall. The MWP and OWP genes are contained in the 9-kilobase-pair (kbp) BclI fragment and constitute an operon under coordinate control of their expression. The nucleotide sequence of a 3.8-kbp EcoRI-SacI fragment containing the entire MWP gene has been determined in this study. Together with the DNA sequence of the promoter region for the MWP-OWP gene operon (H. Yamagata, T. Adachi, A. Tsuboi, M. Takao, T. Sasaki, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 169:1239-1245, 1987) and that of the OWP gene (A. Tsuboi, R. Uchihi, R. Tabata, Y. Takahashi, H. Hashiba, T. Sasaki, H. Yamagata, N. Tsukagoshi, and S. Udaka, J. Bacteriol. 168:365-373, 1986), the complete nucleotide sequence of the MWP-OWP gene operon has been determined. The MWP gene encodes a secretory precursor of the MWP, consisting of a total of 1,053 amino acid residues with a signal peptide of 23 amino acid residues at its amino-terminal end. Bacillus subtilis harboring the MWP gene synthesized an immunoreactive polypeptide with almost the same molecular weight as the authentic MWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid compositions deduced from the MWP and OWP genes were similar to the chemical amino acid compositions of other S-layer proteins in the predominance of acidic amino acids compared with basic amino acids and in the very low content of sulfur-containing amino acids. The acidic nature of the MWP and OWP was confirmed by isoelectric focusing on polyacrylamide gels. In addition, circular dichroism spectra indicated that the S-layer proteins in B. brevis 47 were composed of approximately 30% beta-sheet and 5% alpha-helical structures, with the remainder of the polypeptide backbone being aperiodic in nature.

Amino Acid Sequence↗
PubMed1987

Cloning and characterization of the 5' region of the cell wall protein gene operon in Bacillus brevis 47.

Bacillus brevis 47 secretes vast amounts of proteins derived from both middle wall protein (MWP) and outer wall protein into the medium. The 5' region of the cell wall protein gene operon was cloned into Bacillus subtilis and subsequently into B. brevis 47. On the basis of the nucleotide sequence analysis, an open reading frame coding for MWP was identified on the cloned DNA fragment. Two potential translation initiation sites for the MWP gene are located tandemly in the same reading frame. Each of the sites contains a sequence highly homologous to the 3' end of B. brevis rRNA and an initiation codon. The translational fusion of the 5' region of the MWP gene with the Bacillus licheniformis alpha-amylase gene resulted in the efficient expression of the alpha-amylase gene in B. brevis 47. Of the two potential translation initiation sites, the one located upstream could be eliminated without affecting the expression of the MWP-alpha-amylase fusion gene, suggesting that MWP is synthesized in a precursor form with a signal peptide of 23 amino acid residues. S1 nuclease mapping of the cell wall protein gene transcripts suggested the possibility of the existence of several promoters in the 5' region within 300 base pairs from the translation initiation sites; one promoter was definitely localized within this part of the 5' region, and it was capable of expressing a heterologous gene fusion at a high level. The roles of the apparent structural complexity of the 5' region of the cell wall protein gene operon are discussed in connection with the efficient gene expression.

Amino Acid Sequence↗
PubMed1987

[Cellular responses to multifractionated irradiations].

Several models for dose survival curves in vitro had been proposed by many target, linear quadratic and so on. One of them is a linear quadratic (LQ) model which is described by the equation of E = e-(alpha d + beta d2). Also, the alpha/beta radio for radiation responses of normal or tumor tissues can be determined by their iso-effect relationships. Recently, the studies of LQ model in vitro and in vivo induced to new regimens of radiation therapy: hyperfractionation and superfractionation. The studies for cell growth in multifractionation indicated that the irradiated cells can grow within 4 fractions with daily dose of 2 Gy, thereafter an accumulation of G1 and G2 block were observed. However cell growing fraction seems to be relatively few in the irradiation conditions of more than 3 Gy of daily dose.

Animals↗
PubMed1986

Characterization of the genes coding for two major cell wall proteins from protein-producing Bacillus brevis 47: complete nucleotide sequence of the outer wall protein gene.

Bacillus brevis 47 contains two cell wall proteins termed the outer wall protein (OWP) and middle wall protein (MWP), each of which forms hexagonal arrays in the cell wall. A 6-kilobase BglII-BclI fragment of B. brevis 47 DNA cloned into Bacillus subtilis with a derivative of pHW1 as a vector directed the synthesis of a polypeptide, with almost the same molecular weight as the authentic OWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was specifically recognized by the anti-OWP antibody. Nucleotide sequence analysis of the subfragment revealed that it contains two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the MWP, and the downstream open reading frame corresponds to the entire translational portion of the OWP. The latter encodes a secretory precursor of the OWP, consisting of a total of 1,004 amino acid residues with a signal peptide of 24 amino acid residues at its amino-terminal end. Futhermore, analysis of transcripts in B. brevis 47 suggests that the MWP and OWP genes, in that order, constitute a cotranscriptional unit and that the major promoter shared by the two genes is located upstream of the MWP gene.

Amino Acid Sequence↗