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Biomedical subjects

S Nagata

Publications and source records attributed to S Nagata.

At least 379 records · Page 21Linked to original sources
PubMed1994

Apoptosis regulated by a death factor and its receptor: Fas ligand and Fas.

Homeostasis in vertebrates is tightly regulated by not only proliferation and differentiation of cells, but also cell death or apoptosis (Ellis et al. 1991; Raff 1992). Many cytokines bind to their respective receptors to regulate proliferation and differentiation of cells. Our recent studies on the Fas ligand and Fas indicate that they work respectively as a death factor and its receptor and suggest that, in some cases, cell death or apoptosis is regulated by cytokines and their receptors. Here, I present the summary of the Fas/Fas ligand system which has been studied in my laboratory over the past 5 years, and I will discuss its physiological roles.

Animals↗
PubMed1994

Functional characterization of the promoter for the gene encoding human eosinophil peroxidase.

The molecular basis for commitment of progenitors to the eosinophil lineage and mechanisms by which eosinophil-specific genes are expressed and regulated during differentiation is unknown. Expression of eosinophil peroxidase (EPO) is restricted to the eosinophil lineage. To understand the mechanisms involved in transcriptional regulation of EPO gene expression, we clone the region of the EPO gene upstream of the transcriptional start site and analyzed the cis-acting elements required for EPO promoter activity in an eosinophil-inducible leukemic cell line, HL-60-C15. The 5'-flanking region of the EPO gene containing 1.5 kilobases of sequence upstream of the transcriptional start site was subcloned into the promoterless pXP2-luciferase vector. The EPO-pXP2 construct and 5' deletion mutants were electroporated into HL-60-C15 cells and luciferase reporter activity assessed. The -1.5-kilobase EPO-pXP2 promoter construct reproducibly expressed > 120-fold more luciferase activity than did promoterless pXP2, and a 12-fold (90%) decrease in promoter activity was obtained when sequences between -122 and -45 base pairs (bp) were deleted. The specificity of the EPO promoter for the eosinophil lineage was analyzed by transfecting the EPO-pXP2 constructs and deletion mutants into HL-60-C15 cells and the parental HL-60 line; EPO promoter activity was 8-10-fold less in the HL-60 parental line, suggesting lineage specific elements in the -122 to -45 bp region. To further characterize regulatory sequences important for promoter activity, we performed linker-scanning analysis on the -122 to -45 bp region and identified a number of positively and negatively acting elements in the promoter. DNase I footprinting was performed with HL-60-C15, HL-60, and HeLa nuclear extracts to identify nuclear proteins that may bind to the functional elements; these experiments identified three protected regions of the EPO promoter which correspond to the functional segments defined by linker-scanning analysis and which contain consensus, potential binding sites for Egr-1, H4TF-1, PuF, CTCF, UBP-1, and GaEII transcription factors. Further study of EPO promoter regulation should elucidate unique transcriptional features of eosinophil gene regulation in granulocyte development.

Base Sequence↗
PubMed1994

Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity.

Two molecular mechanisms of T cell-mediated cytotoxicity, one perforin-based, the other Fas-based, have been demonstrated. To determine the extent of their contribution to T cell-mediated cytotoxicity, a range of effector cells from normal control or perforin-deficient mice were tested against a panel of target cells with various levels of Fas expression. All cytotoxicity observed was due to either of these mechanisms, and no third mechanism was detected. Thus, the perforin- and Fas-based mechanisms may account for all T cell-mediated cytotoxicity in short-term in vitro assays.

Amino Acid Sequence↗
PubMed1994

Change in the three-dimensional shape of the stomach in the developing human fetus.

To reveal the developmental characteristics of the fetal stomach in utero, this study was made on 879 subjects from 20 to 40 weeks' gestation. By applying the system of 'Modeling a three-dimensional shape from a silhouette by detecting symmetry' which we devised, we reconstructed the three-dimensional shape, from the outline of the longitudinally transected fetal stomach, obtained from the real-time ultrasound image. Age-related changes in gastric volume and sphericality (parameters produced by the above mentioned method) were analyzed using piecewise linear regression. The fetal stomach volume increased linearly from 20 to 37 weeks' gestation and decreased thereafter to 40 weeks' gestation, with a critical point at 37 weeks' gestation. The sphericality of the fetal stomach shape was also seen to decrease linearly from 20 weeks' to the critical point at 27 weeks' gestation, thereafter remaining constant regardless of the advance in gestation to term. These findings indicate that at 20-27 weeks' gestation, the stomach is of a fusiform-like shape. From 27 weeks' gestation onwards, greater and lesser curvatures become apparent, clearly identifying the cardiac and pyloric regions. The decrease in stomach size after 37 weeks' gestation is also in good accord with the decrease in amniotic fluid volume. The analytical method presented here can help to understand the pathogenesis of gastrointestinal lesions in the developing fetus.

Embryonic and Fetal Development↗
PubMed1994

Identification and characterization of parathyroid hormone/parathyroid hormone-related peptide receptor in cultured astrocytes.

Parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor expressed in rat cultured cortical type I astrocytes was identified and characterized. Northern blot analysis demonstrated that the PTH/PTHrP receptor mRNA is expressed in astrocytes. A human PTH fragment, PTH [1-34], stimulated adenylate cyclase with the EC50 value of 3 nM, but not phospholipase C. PTH [1-34] changed the morphology of protoplasmic type astrocytes into process-bearing ones. Thus, type I astrocytes possess PTH/PTHrP receptor that is coupled to an adenylate cyclase-activating system but not to phospholipase C.

Adenylyl Cyclases↗
PubMed1994

Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.

Mice homozygous for lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) develop lymphadenopathy and suffer from autoimmune disease. The lpr mice have a mutation in a cell-surface protein, Fas, that mediates apoptosis. Fas ligand (FasL) is a tumor necrosis factor (TNF)-related type II membrane protein and binds to Fas. Here, mouse Fasl gene was isolated and localized to the gld region of mouse chromosome 1. Activated splenocytes from gld mice express Fasl mRNA. However, FasL in gld mice carries a point mutation in the C-terminal region, which is highly conserved among members of the TNF family. The recombinant gld FasL expressed in COS cells could not induce apoptosis in cells expressing Fas. These results indicate that lpr and gld are mutations in Fas and Fasl, respectively, and suggest important roles of the Fas system in development of T cells as well as cytotoxic T lymphocyte-mediated cytotoxicity.

Amino Acid Sequence↗
PubMed1994

Structural analysis of the functional gene and pseudogene encoding the murine granulocyte colony-stimulating-factor receptor.

Granulocyte colony-stimulating factor is a cytokine which specifically regulates the production of neutrophilic granulocytes. The granulocyte colony-stimulating-factor receptor (GCSFR) is mainly expressed in neutrophils and their precursor cells. In this study, we isolated the chromosomal gene for murine GCSFR and determined its structure. Like the human GCSFR gene homolog, it consists of 17 exons. The exon-intron organization of the murine and human GCSFR-encoding genes are very similar, except that exon 14 and exon 15 in the murine gene are interrupted by a larger intron (greater than 10 kbp) than that found in the human gene (128 bp). This GCSFR-encoding functional gene (Csfgr) was localized to the distal region of murine chromosome 4 by interspecific backcross mapping. A comparison of the 5' flanking sequence of murine and human Csfgr revealed that a sequence of approximately 300 bp upstream from the cap site is highly conserved. Within this region, an 18-nucleotide element conserved in the promoter of the genes for neutrophil-specific enzymes, was found approximately 140 bp upstream from the cap site, suggesting an involvement of this element in the specific expression of GCSFR in neutrophilic granulocytes. In addition to the functional GCSFR-encoding gene, we isolated a pseudogene for GCSFR, which is flanked by a 15-bp direct repeat at the 5' and 3' ends, and lacks all introns, exons 1-3 and exons 7-8 of the functional gene. The processed pseudogene has, in its most 5' region, a sequence of approximately 200 bp that is highly related to the DNA sequence approximately 1.2 kbp upstream of the cap site of the functional gene.

Animals↗
PubMed1994

Purification and characterization of the Fas-ligand that induces apoptosis.

Fas is a 45-kD cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family, and transduces the signal for apoptosis. The cytotoxic T lymphocyte (CTL) hybridoma, PC60-d10S requires the presence of Fas on target cells to induce cytolysis in target cells. This CTL cell line was weakly but specifically stained by a chimeric protein that consisted of the extracellular domain of mouse Fas and the Fc portion of human immunoglobulin G1 (mFas-Fc). Moreover, mFas-Fc inhibited the cytotoxic activity of PC60-d10S. Sublines of d10S that were stained intensively by mFas-Fc were isolated by repetitive fluorescence-activated cell sorter sorting. A cell-surface protein of about 40 kD was specifically precipitated by mFas-Fc from the lysates of these sublines. This protein was homogeneously purified by sequential affinity chromatographies using mFas-Fc and concanavalin A beads. The purified protein exhibited cytotoxic activity against cells expressing Fas but not to the cells which do not express Fas. These results indicated that the 40-kD membrane glycoprotein expressed on PC60-d10S cells is the Fas-ligand that induces the apoptotic signal by binding to Fas.

Animals↗
PubMed1994

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO and CD3 epsilon in a CD7+ CD5- CD2- case MPO, CD3 epsilon, and CD3 delta in a CD7+ CD5+ CD2- case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during immature stages of differentiation. This helps understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML).(ABSTRACT TRUNCATED AT 400 WORDS)

Acute Disease↗
PubMed1994

Influence of intraballoon pressure on development of severe mitral regurgitation after percutaneous transvenous mitral commissurotomy.

To evaluate the influence of intra-balloon pressure on the development of severe mitral regurgitation (> or = grade 3+), we measured intraballoon pressure during percutaneous transvenous mitral commissurotomy (PTMC) in 62 patients using the Inoue balloon catheter. The peak intraballoon pressure was 2.29 +/- 0.55 kg/cm2. Severe mitral regurgitation as a result of leaflet tear occurred in 7 patients (11%). Patients were divided into two groups those with (n = 7) and those without (n = 55) severe mitral regurgitation. Intraballoon pressure had been significantly higher in those with vs. those without severe mitral regurgitation (2.76 +/- 0.31 kg/cm2 vs. 2.23 +/- 0.55 kg/cm2, P < 0.01). Multiple logistic regression analysis revealed that the occurrence of severe mitral regurgitation was related to only the peak intraballoon pressure. These data suggest that a high intraballoon pressure is a risk factor for severe mitral regurgitation as a result of leaflet tear.

Adult↗
PubMed1994

Increased Fas antigen expression in murine retrovirus-induced immunodeficiency syndrome, MAIDS.

The Fas antigen (Fas), which is a cell surface protein belonging to the tumor necrosis factor receptor family, mediates apoptosis. To assess the contribution of Fas to the pathogenesis of retrovirus-induced immunodeficiency, we examined the kinetics of Fas expression on the lymphocytes during the course of murine acquired immunodeficiency syndrome (MAIDS) induced by a defective LP-BM5 murine leukemia virus. The Fas-positive cells were increased in proportion both in alpha beta T cells and B cells with the progression of MAIDS. The appearance of Fas-positive cells in alpha beta T cells preceded those in B cells during the course of MAIDS. Among alpha beta T cells, about half of the Thy1.2+ alpha beta T cells were positive for Fas, while almost all of Thy1.2- CD4+ alpha beta T cells were of the Fas-positive phenotype. The Fas-positive cells in MAIDS mice, especially unique Thy1.2-CD4+ alpha beta T cells, were easily rendered apoptotic by stimulation via Fas, indicating that Fas expressed on the lymphocytes is functional. Furthermore, concomitant infection with Mycobacterium avium in MAIDS mice caused a marked increase in Fas-positive cells accompanied by a severely impaired T cell reactivity to polyclonal stimuli. Taken together, these results suggest that possible participation of the Fas system in the pathogenesis of retrovirus-induced immunodeficiency.

Animals↗
PubMed1994

Production of human granulocyte colony stimulating factor by various kinds of stromal cells in vitro detected by enzyme immunoassay and in situ hybridization.

Production of human granulocyte colony stimulating factor (G-CSF) by stromal cells was studied in vitro. Induction of G-CSF by interleukin 1 (IL-1) and lipopolysaccharide (LPS) was compared using enzyme immunoassay in various kinds of stromal cells. Primary human bone marrow stromal cells, a human bone marrow-derived stromal cell line (KM-102), and peripheral blood monocytes secreted small amounts of G-CSF without stimulation, while vascular endothelial cells and skin fibroblasts secreted G-CSF only when induced by IL-1 or LPS. The production of G-CSF by monocytes was stimulated predominantly by LPS, whereas that by KM-102 cells, endothelial cells, and fibroblasts was induced by IL-1 but much less so by LPS. IL-1 and LPS stimulated similar levels of G-CSF production by primary bone marrow stromal cells which consisted of various types of cells. In situ hybridization for G-CSF mRNA showed that only a small proportion of primary bone marrow stromal cells expressed a large amount of G-CSF mRNA upon stimulation. The positive cells were round or oval in shape, while most of the spindle-shaped stromal cells were negative for specific grains. Although further characterization of positive cells is needed, the results suggest that bone marrow stromal cells are heterogeneous in terms of their capacity for G-CSF production.

3T3 Cells↗
PubMed1994

Fas-mediated apoptosis in primary cultured mouse hepatocytes.

The Fas antigen is a member of the tumor necrosis factor/nerve growth factor receptor family and is expressed in tissues such as the thymus, liver, and ovary. Agonistic anti-Fas antibodies have cytolytic activity against cell lines expressing the Fas antigen. In this study, we show that anti-Fas antibody can induce the death of mouse hepatocytes in primary culture. Cell death via apoptosis was evidenced by the fact that the dying cells displayed DNA fragmentation, extensive surface bleb formation, and chromatin condensation. Anti-Fas antibody alone induced apoptosis in less than 20% of the cultured hepatocytes, whereas all cells were killed within 24 h by anti-Fas antibody in the presence of actinomycin D, cycloheximide, or protein kinase C (PKC) inhibitors such as H-7 and HA1004. These results indicate that the Fas antigen expressed in mouse hepatocytes functionally transduces the apoptotic signal and suggest that cultured mouse hepatocytes express protective proteins against apoptosis and that phosphorylation by PKC is also involved in protection of the hepatocytes from Fas-mediated apoptosis.

Animals↗
PubMed1994

Mutations in the Fas antigen gene in lpr mice.

The Fas antigen is a cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family and it mediates apoptosis. The Fas antigen gene is the structural gene for the mouse mutation, lpr (lymphoproliferation). In one allele of lpr, an early transposable element (a mouse endogenous retrovirus) is inserted into an intron of the Fas antigen gene, which causes premature termination and aberrant splicing of the Fas antigen transcript. In the other allele (lprcg), a point mutation in the signal transducing domain of the cytoplasmic region abolishes the function of the Fas antigen.

Animals↗
PubMed1994

Total en bloc spondylectomy for solitary spinal metastases.

We have developed a technique for total en bloc spondylectomy through a posterior approach and now report our experience of 20 patients with a solitary or localised metastasis in the thoracic or lumbar vertebrae. There are two steps: an en bloc laminectomy, followed by en bloc resection of the vertebral body with an oncological wide margin and the insertion of a vertebral prosthesis. Pain was relieved in the 17 patients who could be assessed; 11 of the 15 patients with a neurological deficit were much improved, impending paralysis being prevented in 5 patients. There have been no local recurrences. Nine patients are at present alive with a mean follow up of 17.4 months.

Adult↗
PubMed1994

Diastolic suction in the human ventricle: observation during balloon mitral valvuloplasty with a single balloon.

Diastolic suction has been demonstrated experimentally as a ventricular negative pressure when the ventricle is allowed to relax completely in the absence of filling, but it has not been extensively studied in the in vivo human heart. In balloon mitral valvuloplasty with a single balloon, the mitral orifice is occluded and inflow is considered to be completely obstructed during a balloon inflation. To demonstrate diastolic suction in the human ventricle, we measured left ventricular pressure during valvuloplasty with a high-fidelity catheter tip manometer in 17 patients. Left ventricular pressure fell below zero during a balloon inflation in all patients (-2 to -12 mm Hg). The peak negative diastolic pressure showed significant correlations with end-systolic volume index (r = 0.53, p = 0.03) and with the ejection fraction (r = 0.80, p = 0.0001). Thus diastolic suction was demonstrated in the human beating heart, and the sucking effect was potent in the heart with small end-systolic volume and high-ejection fraction.

Adult↗