PubMed Health⌕ Search

Biomedical subjects

A Tsuboi

Publications and source records attributed to A Tsuboi.

At least 55 records · Page 3Linked to original sources
PubMed1995

[RBE of carbon beam on hematopoietic injury].

The inactivation of hematopoiesis and survival of mice have been used in determining the relative biological effectiveness (RBE) of heavy charged particles of carbon 12 generated by Riken ring-cyclotron. Whole bodies of anesthetized mice were exposed to accelerator-generated carbon 12 of 135 MeV/u or X-rays of 200 kVp in graded doses. At about 5 hours after exposure, exogenous spleen colonies (CFU-S), granulocyte-macrophage progenitor cells (GM-CFC) and megakaryocyte progenitor cells (Meg-CFC) were assayed to determine dose relationships, and then D0 doses were determined. The inactivation of blood cells and lethality of mice (LD50/30) were also monitored for 30 days after irradiation. The RBE values of the 135 MeV/u carbon beam in terms of CFU-S were 1.68 for marrow and 1.41 for spleen, for GM-CFC they were 1.56 for marrow and 3.29 for spleen, for Meg-CFC they were 1.25 for marrow and 3.73 for spleen, while they were about 1.0 for erythrocytes, leukocytes and thrombocytes in mice irradiated with 2 Gy. However, the RBE values of the carbon beam were 1.32 for LD50/30.

Animals↗
PubMed1994

The transcription factor Sp1 is required for induction of the murine GM-CSF promoter in T cells.

The cis-acting region, GM-kappa B/GC-box (positions -95 and -73), within the murine GM-CSF gene promoter is required for maximal induction by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in T cells. GM-kappa B defines a binding site for NF-kappa B, and GC-box defines a binding site for three (A1, A2, B) constitutive proteins. We report here that three copies of the GC-box can functionally compensate for the GM-kappa B/GC-box region, suggesting that the GC-motif can function independently of the GM-kappa B motif. The major GC-box binding activity A1 was purified and identified as the transcription factor Sp1. We show that depletion of Sp1 (A1) from nuclear extracts specifically decreases in vitro transcription activity on GM-CSF templates. Since the human GM-CSF promoter has a base difference within the GC-box, we speculate that this may explain why the human promoter is weak and that an upstream enhancer is required for the induction of the human GM-CSF gene.

Animals↗
PubMed1994

Differential response of NF-kappa B1 p105 and NF-kappa B2 p100 to HTLV-I encoded Tax.

In a previous study we found that HTLV-I encoded Tax transactivator, which binds to NF-kappa B1 p105, suppresses p105-mediated I kappa B activity, thereby allowing entry of NF-kappa B p65 (RelA) and p50 into the nucleus. In the present report, we compared the effect of Tax on NF-kappa B2 p100, which also binds to Tax, with that of p105 in transfected COS7 cells. While p105 is processed to the DNA binding form, p50, processing of p100 was much less efficient both in the presence and absence of Tax. Both p105 and p100 showed I kappa B activity in sequestering NF-kappa B p65 into the cytoplasm. However, only p105-mediated I kappa B activity, and not that of p100, was inhibited by Tax. Chimeric molecules between p100 and p105 suggested that inefficient processing of p100 can be attributed to the Rel homologous domain, rather than to the ankyrin repeat domain. p100, but not p105, potently suppressed Tax- and p65-induced transaction of a GM-CSF promoter in Jurkat cells. Taken together, these results suggest that p105 and p100 may have distinct effects on Tax-induced transactivation events.

Amino Acid Sequence↗
PubMed1994

Characterization of NF(P), the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to NF-kappa B and NF-AT.

The P sequence of the human interleukin-4 (IL-4) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the NF-kappa B and the NF-AT binding sites. We examined whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and NF-AT. NF-kappa B (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, NF(P) binding was inhibited by the unlabeled NF-AT binding site but not by the unlabeled AP1 binding site and purified NF-AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of NF-AT were similar to those of NF(P) and only a small amount of P65 was detected in NF(P) in crude nuclear extracts. These results indicate that the component(s) of NF-AT has the potential to reconstitute NF(P) whereas NF-kappa B alone cannot account for NF(P) in crude extracts. Unlike NF-AT, NF(P) does not contain AP1 as its DNA binding component.

Base Sequence↗
PubMed1994

Calcineurin activates transcription from the GM-CSF promoter in synergy with either protein kinase C or NF-kappa B/AP-1 in T cells.

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)-expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+-signaling pathways downstream of T cell activation.

Animals↗
PubMed1994

Molecular basis for developmental changes of GM-CSF gene inducibility in embryonal carcinoma cells.

In a previous report, we reported that the induction of GM-CSF gene in differentiated P19 cells results from the maturation of the transcriptional machinery. Here, we identified a cis-DNA element which confers the activation of GM-CSF gene in response to PMA/A23187 stimulation in differentiated state. Analysis of the 5'-flanking region between -113 and -60 revealed two elements responsible for promotion and one for inhibition, and the overall effects led to the activation of GM-CSF gene mainly through the sequence between -95 and -73. Using the oligonucleotide between -94 and -73 as a probe in gel retardation assays, we identified a DNA-binding protein, NF-GM-P19, the binding activity of which was induced after differentiation in response to PMA/A23187 stimulation. These results indicate that the induction of GM-CSF gene after differentiation results from the maturation of the transcriptional machinery which recognizes the sequence between -95 and -73.

Animals↗
PubMed1994

Developmental changes of GM-CSF gene inducibility in embryonal carcinoma cells.

Murine embryonal carcinoma (EC) P19 cells, a tissue culture model of early embryonic development, failed to produce cytokines, such as interleukin-3 (IL-3), IL-4, granulocytemacrophage colony stimulating factor (GM-CSF) and interferon-beta (IFN-beta) at the mRNA level. Differentiation induced by retinoic acid (RA) released this repression to produce some cytokines. GM-CSF and IFN-beta genes were expressed in response to PMA/A23187, poly(I):poly(C), IL-1 alpha, forskolin, or LPS stimulation in differentiated P19 cells, whereas IL-3 and IL-4 genes were not expressed. To elucidate the mechanism of the GM-CSF gene induction after differentiation, we transfected a series of 5' deletion mutants of the mouse GM-CSF promoter fused to the bacterial CAT gene. The 740-bp fragment of the 5'-flanking region mediated the positive response. Deletion analysis revealed that the 5' boundary region of the DNA element required for activation lies between positions -95 and -84 and the region upstream of position -95 appears inhibitory. These results indicate that the maturation of the transcriptional machinery after differentiation results in the activation of the GM-CSF gene.

Animals↗
PubMed1994

Calcineurin potentiates activation of the granulocyte-macrophage colony-stimulating factor gene in T cells: involvement of the conserved lymphokine element 0.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

Animals↗
PubMed1993

Purification of the 120 kDa component of the human nuclear factor of activated T cells (NF-AT): reconstitution of binding activity to the cis-acting element of the GM-CSF and IL-2 promoter with AP-1.

The cis-acting element of the granulocyte-macrophage colony-stimulating factor (GM-CSF) promoter, CLE0, is required for stimulation dependent expression of the GM-CSF gene by phorbol ester (PMA) and calcium ionophore (A23187) in T cells. We recently obtained evidence that NF-CLE0 gamma, one of the CLE0-binding factors, is similar to the nuclear factor of activated T cells, NF-AT. In the present study, we show that the affinity-purified NF-AT from nuclear extracts of human Jurkat T cells stimulated with both PMA and A23187 bound strongly to the CLE0 element and formed a NF-CLE0 gamma complex. This DNA-protein complex was competitively inhibited by oligonucleotides containing NF-AT and AP-1 binding sites, suggesting that the CLE0 gamma complex is identical to NF-AT and contains AP-1 proteins. Here, one component of NF-AT with an apparent molecular mass of 120 kDa on SDS-polyacrylamide gel electrophoresis was purified to near homogeneity by Mono Q chromatography. The purified 120 kDa protein reconstitutes NF-AT binding in combination with recombinant cJun/cFos heterodimer. Furthermore, we demonstrate that binding of this 120 kDa protein to both the NF-AT and the CLE0 sequences can be reconstituted with the addition of affinity-purified Jurkat AP-1 proteins. These results indicate that NF-AT (NF-CLE0 gamma), which is composed of the 120 kDa nuclear protein and AP-1 proteins, regulates the stimulation-dependent expression of the GM-CSF gene as it does the IL-2 gene.

Base Sequence↗
PubMed1993

The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT.

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.

Animals↗
PubMed1993

[Pulmonary venous obstruction after repair of total anomalous pulmonary venous drainage: report of a case with reoperation].

A 8-month-old infant with Darling' type Ib of total anomalous pulmonary venous drainage underwent surgical correction. Common pulmonary vein drained into superior vena cava. Pulmonary venous channel was constructed between orifice of common pulmonary vein and atrial septal defect (ASD) with pericardial baffle drainage. Pulmonary venous obstruction occurred at the site of ASD and pericardial baffle was atrophic. ASD was enlarged and new pulmonary venous channel was reconstructed with EPTFE graft. Post-reoperative clinical course was uneventful.

Female↗
PubMed1992

The carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial sigma factors and is required for function.

Transcription factor beta gamma (RAP30/74) from rat liver was previously shown in biochemical studies to control the binding of RNA polymerase II to promoters by a mechanism analogous to that utilized by bacterial sigma factors, by decreasing the affinity of polymerase for nonpromoter sites on DNA and by increasing the affinity of the enzyme for the preinitiation complex (Conaway, R. C., Garrett, K. P., Hanley, J. P., and Conaway, J. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6205-6209). By constructing and analyzing mutants of beta gamma, we have identified a novel functional domain located in the carboxyl terminus of the gamma (RAP30) subunit. This domain shares sequence similarity with region 4 of bacterial sigma factors; in particular, it exhibits striking similarity to the carboxyl-terminal regions 4.1 and 4.2 of SpoIIIC (Bacillus subtilis sigma k). Evidence from biochemical studies argues that a mutant gamma (RAP30), lacking amino acid sequences similar to sigma homology region 4.2, is able to assemble with the beta (RAP74) subunit to form a mutant beta gamma (RAP30/74) with impaired ability to interact with RNA polymerase II.

Amino Acid Sequence↗
PubMed1992

Activation of lymphokine genes in T cells: role of cis-acting DNA elements that respond to T cell activation signals.

Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Th1 and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines. The production of the granulocyte-macrophage colony stimulating factor (GM-CSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions -95 to +27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein(s) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLEO defines a binding site for protein(s) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-1, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation.

Animals↗
PubMed1992

Protection of survival and hematopoiesis in irradiated mice by 5-fluorouracil.

The effects of whole-body irradiation on survival and the hematopoietic system were studied in mice treated with 5-fluorouracil (5-FU). Animals (ddY-SLC male mice, 8-10 weeks old) were injected with 5-FU (i.p.) as a single dose (150 mg/kg) at various times before or after irradiation with X-rays at graded doses (4.8 to 7.6 Gy). The treatment of mice with 5-FU 5 days before irradiation was the most effective for the reduction of radiation lethality, having a radioprotective effect. The dose reduction factor (DRF) was 1.24. However, treatment with 5-FU at 1 day and 2 hours before, or at various times after irradiation significantly increased the radiation lethality compared to the untreated controls, creating a radiosensitizing effect. The decrease or the increase of radiation lethality exhibited by 5-FU was similar to the radiation-dose relationship pattern shown by endogenous and exogenous CFU-S. The pattern of change of thrombocyte counts in the circulating blood after irradiation was greatly modified by pretreatment with 5-FU 5 days before irradiation, effectively lessening the radiation-induced depression. In contrast, the post-irradiation patterns of leukocyte and erythrocyte variation did not show any significant change due to pretreatment with 5-FU.

Animals↗
PubMed1992

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.

Amino Acid Sequence↗