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S Inoue

Publications and source records attributed to S Inoue.

At least 541 records · Page 30Linked to original sources
PubMed1997

Dihydro-1,4-benzothiazine-6,7-dione, the ultimate toxic metabolite of 4-S-cysteaminylphenol and 4-S-cysteaminylcatechol.

4-S-Cysteaminylphenol (4-S-CAP) and the corresponding catechol 4-S-cysteaminylcatechol (4-S-CAC) have been evaluated for melanocytotoxicity. It was shown recently that tyrosinase oxidation of these substrates produces a violet pigment, dihydro-1,4-benzothiazine-6,7-dione (BQ). In this study we examined whether BQ is the ultimate toxic metabolite produced in melanoma cells from 4-S-CAP/4-S-CAC. Biochemical experiments showed that (1) BQ was formed by autoxidation of 4-S-CAC as well as by tyrosinase oxidation of 4-S-CAP/4-S-CAC, (2) BQ reacted rapidly with thiols such as reduced glutathione (GSH), and (3) BQ inhibited the activity of alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that (1) the cytotoxicity of 4-S-CAC was mostly prevented by catalase and superoxide dismutase, (2) BQ was highly cytotoxic to B16 melanoma cells (IC50 being 3.9 microM as compared with 507 microM for 4-S-CAP), (3) BQ was metabolized rapidly to a GSH adduct in melanoma cells, and (4) the same GSH adduct was also formed upon incubation of melanoma cells with 4-S-CAP, the reaction being tyrosinase dependent. In vivo experiments showed that intratumoral administration of BQ (0.5 micromol) inhibited the subcutaneous growth of B16 melanoma nearly as effectively as 4-S-CAP/4-S-CAC (20 micromol). These results indicate that BQ is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-S-CAP/4-S-CAC. BQ deprives melanoma cells of GSH and may inactivate SH enzymes essential for DNA synthesis and cell proliferation by covalent binding through their cysteine residues, thereby exerting melanocytotoxicity. Cytotoxicity of 4-S-CAC depends mostly on autoxidation producing BQ and active oxygens.

Animals↗
PubMed1997

BRCA2 germline mutations in Japanese breast cancer families.

Germline mutations of BRCA2 were examined in 20 Japanese breast cancer families without BRCA1 mutations, including one demonstrating cancer development in a male. Three different mutations, resulting in truncation of the BRCA2 protein, were detected in 3 different families. They were 9474insA (exon 24, termination at codon 3110), C8729A (exon 20, S2834 ter) and 982del4 (exon 9, termination at codon 275). The 982del4 mutation was detected in the family with a case of male breast cancer. Age at onset was young, with a range of 28-43 years, in the 2 female breast cancer families with truncation mutations. One probable missense mutation, A10462G (13412V), was further detected in 2 families, although cosegregation of this allele with the breast cancer phenotype was not complete. The rate of BRCA2 mutations in Japanese families was suggested to be almost the same as in Western countries, and larger than it is the case for BRCA1.

Adult↗
PubMed1997

Functional interaction between DNA-PK and c-Abl in response to DNA damage.

How DNA damage is converted into intracellular signals that can control cell behaviour is unknown. The c-Abl protein tyrosine kinase is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of a serine/threonine kinase and Ku DNA-binding subunits, requires DNA double-strand breaks or other DNA lesions for activation. Here we demonstrate that c-Abl interacts constitutively with DNA-PK. Ionizing radiation stimulates binding of c-Abl to DNA-PK and induces an association of c-Abl with Ku antigen. We show that DNA-PK phosphorylates and activates c-Abl in vitro. Cells deficient in DNA-PK are defective in c-Abl activation induced by ionizing radiation. In a potential feedback mechanism, c-Abl phosphorylates DNA-PK, but not Ku, in vitro. Phosphorylation of DNA-PK by c-Abl inhibits the ability of DNA-PK to form a complex with DNA. We also show that treatment of cells with ionizing radiation results in phosphorylation of DNA-PK that is dependent on c-Abl. Our results support the hypothesis that there are functional interactions between c-Abl and DNA-PK in the response to DNA damage.

Animals↗
PubMed1997

Identification of sulfated oligosialic acid units in the O-linked glycan of the sea urchin egg receptor for sperm.

The Strongylocentrotus purpuratus sea urchin egg receptor for sperm is a cell surface glycoprotein with a molecular mass of 350 kDa. Recent studies indicate that the sulfated O-linked glycans isolated from the receptor bind to acrosome-reacted sperm. The purified receptor was analyzed with respect to amino acid and carbohydrate content and shown to be composed of 70% carbohydrate by weight. Compositional analysis indicated that both N- and O-linked oligosaccharide chains were present. After peptide:N-glycanase treatment of the receptor to remove most of the N-linked glycan chains, the majority of the sialic acid residues remained associated with the receptor and were shown by several types of experiments to be composed of sulfated oligosialic acid units attached to the O-linked glycan chains of the receptor. Chemical and physical studies on oligosialic chains discovered earlier in the Pronase-generated glycopeptide fraction isolated from the egg cell surface complex of another species of sea urchin, Hemicentrotus pulcherrimus, established that these molecules had the structure: (SO(4)-)-9Neu5Gc alpha2(-->5-O(glycolyl)Neu5Gc alpha2-->)n. Based on comparative and analytical studies, it was concluded that this sulfated oligosaccharide is a component of a GalNAc-containing chain that is O-linked to the polypeptide chain of the sea urchin egg receptor for sperm. Using a competitive inhibition of fertilization bioassay it was shown that the sulfated oligosialic acid chains derived from the S. purpuratus egg cell surface complex inhibited fertilization; the nonsulfated form of this oligosialic chain had little inhibitory activity.

Animals↗
PubMed1997

IL-2-induced IL-5 synthesis, but not proliferation, of human CD4+ T cells is suppressed by FK506.

Regulation of T cell IL-5 synthesis was investigated using human Th cell clones. Immunosuppressant FK506 suppressed IL-5 synthesis of T cells activated through TCR in a dose-dependent manner. IL-5 gene transcription and protein synthesis were also induced in the same T cell clones upon stimulation with IL-2 and were suppressed by FK506 in a dose response similar to that induced by TCR stimulation. In contrast to TCR stimulation, neither activating protein-1, nuclear factor-AT (NF-AT), nor NF-kappaB binding activity was significantly up-regulated by IL-2 stimulation. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T cell clones was transcribed upon either TCR or IL-2 stimulation and was clearly down-regulated by FK506, indicating that the approximately 500-bp human IL-5 gene segment located 5' upstream of the coding region contained FK506-sensitive enhancer elements. Our present findings clearly indicate that FK506-sensitive signaling molecules are involved in T cell IL-5 production induced by both TCR and IL-2 stimulation and suggest that IL-2 receptor signal leading to IL-5 gene transcription is transduced by a unique FK506-sensitive pathway other than the Ca2+-dependent signal transduction pathway, such as the calcineurin-NF-AT system.

CD4-Positive T-Lymphocytes↗
PubMed1997

Two distinct pathways of interleukin-5 synthesis in allergen-specific human T-cell clones are suppressed by glucocorticoids.

Glucocorticoids (GC) have long been used as the most effective agents for the treatment of allergic diseases accompanied by eosinophilia such as chronic asthma and atopic dermatitis. The development of chronic eosinophilic inflammation is dependent on interleukin-5 (IL-5), a selective eosinophil-activating factor, produced by helper T cells. To delineate the regulatory mechanisms of human IL-5 synthesis, we established allergen-specific CD4+ T-cell clones from asthmatic patients. GC efficiently suppressed IL-5 synthesis of T-cell clones activated via either T-cell receptor (TCR) or IL-2 receptor (IL-2R). Induction of IL-5 mRNA upon TCR and IL-2R stimulation was totally inhibited by dexamethasone. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T-cell clones was transcribed on either TCR or IL-2R stimulation and was clearly downregulated by dexamethasone, indicating that the approximately 500-bp human IL-5 gene segment located 5' upstream of the coding region contains activation-inducible enhancer elements responsible for the regulation by GC. Electrophoretic mobility shift assay analysis suggested that AP-1 and NF-kappaB are among the possible targets of GC actions on TCR-stimulated T cells. NF-AT and NF-kappaB were not significantly induced by IL-2 stimulation. Our results showing that GC suppressed IL-5 production by human CD4+ T cells activated by two distinct stimuli, TCR and IL-2R stimulation, underscore the efficacy of GC in the treatment of allergic diseases via suppression of T-cell IL-5 synthesis.

Allergens↗
PubMed1997

Expressed emotion among families and course of schizophrenia in Japan: a 2-year cohort study.

A 2-year cohort study was conducted to determine whether expressed emotion (EE) in families is associated with relapse of schizophrenia in Japan. Key relatives of 52 schizophrenic patients were interviewed using the Camberwell Family Interview (CFI) and the patients were classified into high and low EE groups. The 2-year relapse risks were 71% (17/24) in the high EE group and 37% (10/27) in the low EE group. The risk ratio was 1.9 and the 95% confidence interval (CI) ranged from 1.1 to 3.3. In the multiple logistic regression analysis, the odds ratio between families' EE and schizophrenic relapse was 4.6, controlling for other potential confounding factors. It was concluded that, in Japan, the families' EE is a good predictor for schizophrenic relapse within 2 years. This association between EE and relapse could be due to the strong difference in the first 9 months. The period during which EE's predictive validity for schizophrenic relapse is applicable may vary according to the society to which the patients and their families belong.

Adolescent↗
PubMed1997

The brain finger protein gene (ZNF179), a member of the RING finger family, maps within the Smith-Magenis syndrome region at 17p11.2.

Smith-Magenis syndrome (SMS) is caused by a microdeletion of 17p11.2 and comprises developmental and growth delay, facial abnormalities, unusual behavior and sleep problems. This phenotype may be due to haploinsufficiency of several contiguous genes. The human brain finger protein gene (ZNF179), a member of the RING finger protein family, has been isolated and mapped to 17p11.2. FISH analyses of metaphase or interphase chromosomes of 6 patients with SMS show that ZNF179 was deleted in one of the 2 homologs (17p11.2), indicating a possible association of the defect of this gene with the pathogenesis of SMS. Furthermore, using a prophase FISH ordering system, we sublocalized ZNF179 proximally to LLGL which lies on the critical region for SMS.

Abnormalities, Multiple↗
PubMed1997

Catalysis by a new sialidase, deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm), initially forms a less stable alpha-anomer of 3-deoxy-D-glycero-D-galacto-nonulosonic acid and is strongly inhibited by the transition state analogue, 2-deoxy-2, 3-didehydro-D-glycero-D-galacto-2-nonulopyranosonic acid, but not by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid.

Deaminoneuraminic acid residue-cleaving enzyme (KDNase Sm) is a new sialidase that has been induced and purified from Sphingobacterium multivorum. Catalysis by this new sialidase has been studied by enzyme kinetics and 1H NMR spectroscopy. Vmax/Km values determined for synthetic and natural substrates of KDNase Sm reveal that 4-methylumbelliferyl-KDN (KDNalpha2MeUmb, Vmax/Km = 0.033 min-1) is the best substrate for this sialidase, presumably because of its good leaving group properties. The transition state analogue, 2, 3-didehydro-2,3-dideoxy-D-galacto-D-glycero-nonulosonic acid, is a strong competitive inhibitor of KDNase Sm (Ki = 7.7 microM versus Km = 42 microM for KDNalpha2MeUmb). 2-Deoxy-2, 3-didehydro-N-acetylneuraminic acid and 2-deoxy-2, 3-didehydro-N-glycolylneuraminic acid are known to be strong competitive inhibitors for bacterial sialidases such as Arthrobacter ureafaciens sialidase; however, KDNase Sm activity is not significantly inhibited by these compounds. This observation suggests that the hydroxyl group at C-5 is important for recognition of the inhibitor by the enzyme. Reversible addition of water molecule (or hydroxide ion) to the reactive sialosyl cation, presumably formed at the catalytic site of KDNase Sm, eventually gives rise to two different adducts, the alpha- and beta-anomers of free 3-deoxy-D-glycero-D-galacto-nonulosonic acid. 1H NMR spectroscopic studies clearly demonstrate that the thermodynamically less stable alpha-form is preferentially formed as the first product of the cleavage reaction and that isomerization rapidly follows, leading to an equilibrium mixture of the two isomers, the beta-isomer being the major species at equilibrium. Therefore, we propose that KDNase Sm catalysis proceeds via a mechanism common to the known exosialidases, but the recognition of the substituent at C-5 by the enzyme differs.

Flavobacterium↗
PubMed1997

Activity of UDP-GlcNAc:GlcNAc beta 1-->6(GlcNAc beta 1-->2) Man alpha 1-->R[GlcNAc to Man] beta 1-->4N-acetylglucosaminyltransferase VI (GnT VI) from the ovaries of Oryzias latipes (Medaka fish).

UDP-GlcNAc:GlcNAc beta 1-->(GlcNAc beta 1-->2)Man alpha 1-R[GlcNAc to Man] beta 1-->4N-acetylglucosaminyltransferase VI (GnT VI) activity was shown to be present in crude homogenates of Medaka fish (Oryzias latipes) ovaries using UDP-[14C]GlcNAc and synthetic GlcNAc beta 1-->6 (GlcNAc beta 1-->2)Man alpha 1-->6Glc beta 1-->octyl as substrates. Characterization of this activity showed a pH optimum at about pH 7.0 and an absolute requirement for divalent cations. The optimum concentration of Mn2+ was at about 25 mM. This finding is the first report on GnT VI activity in fish; the enzyme has previously been described only in avian tissues.

Animals↗
PubMed1997

Mapping of the catalytic and epitopic sites of human CD38/NAD+ glycohydrolase to a functional domain in the carboxyl terminus.

We reported that 1) ecto-NAD+ glycohydrolase (NADase) activity induced upon differentiation of HL-60 cells is localized on the extracellular carboxyl-terminal side of CD38 and that 2) CD38 ligation by specific mAbs is followed by protein tyrosine phosphorylation in the cells. The strategy selected for identifying the relevant catalytic domains of the molecule relies upon the production in COS-7 cells of carboxyl-terminal deletion mutants of CD38. The mutants with fewer than 15 amino acids deleted at the carboxyl terminus of the 300-amino acid wild-type molecule maintained NADase activity, whereas those with more than 27 amino acids deleted did not. The general inference is that the carboxyl-terminal 273-285 sequence bears the site of enzyme activity. Introduction of site-directed mutation of a conserved cysteine residue (Cys275), located in the 273-285 sequence, completely abolished NADase activity. The second issue resolved in this work is the definition of an epitope of the agonistic anti-CD38 mAbs. To this aim, a panel of selected anti-CD38 mAbs was tested using these mutants and various CD38 fragments as the target in immunoblot analyses. All of the epitopes recognized by mAbs inducing protein tyrosine phosphorylation were mapped on an identical site containing the carboxyl-terminal sequence of 273-285. The conclusion is that the discrete carboxyl-terminal sequence identified in the present study not only plays a key role in its ecto-NADase activity, but actually constitutes the epitopes exploited by the agonistic anti-CD38 mAbs for transmembrane signaling.

ADP-ribosyl Cyclase↗
PubMed1997

In vitro proliferation and differentiation of erythroid progenitors of cord blood.

Stem cell factor (SCF) is known to synergize with erythropoietin (EPO) for erythropoiesis in vitro. Clonogenic assay and suspension culture were used to assess the effect of EPO alone or its combination with SCF on the proliferation and differentiation of erythroid progenitors of cord blood. Colony formation, increase in cell count, and cell cycling status for the proliferation as well as expression of Glycophorin A (Gly A) and hemoglobinization as the marker of differentiation were determined with each stimulation. The cell cycle status of the cells in suspension cultures was determined using FACScan after labeling of cells with propidium iodide. Expression of Gly A and degree of hemoglobinization were determined by FACScan and spectrophotometer on the cells plucked from colonies in semisolid culture. Larger increases in cell counts in suspension culture were observed with EPO + SCF after 12 days of inoculation than with EPO alone. Mean doubling time was 14.2 h with EPO + SCF and 22.7 h with EPO alone. The proportion of cells in S and G2 + M phase in day 14 suspension culture was 48% with EPO + SCF and 43% with EPO alone (no significant difference). Mean colony counts per 10(5) nonadherent mononuclear cells were 76 +/- 14 with EPO + SCF and 51 +/- 15 with EPO at day 14 (p < 0.05). The number of macroscopic colonies with > 0.5 mm diameter was 10.7 +/- 1.2 with EPO + SCF and 0.3 +/- 0.5 with EPO (p < 0.05). Percent of Gly A+ cells was 75% for both EPO + SCF and EPO colonies at day 14. Hemoglobin concentration/10(5) cells at day 14 was 0.70 +/- 0.17 microgram with EPO + SCF, and 1.16 +/- 0.32 micrograms with EPO alone (p < 0.05). In conclusion, SCF in the combination with EPO showed a synergistic effect for erythroid proliferation in colony number as well as colony size derived from cord blood, while SCF with EPO decreased hemoglobin synthesis but not Gly A expression at day 14.

Blood Cell Count↗
PubMed1997

Signal transduction via the CD38/NAD+ glycohydrolase.

The human cell surface CD38 molecule is a 46-kDa type-II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We previously demonstrated that an ecto-form NAD+ glycohydrolase (NADase) activity induced by all-trans retinoic acid in HL-60 cells is due to the extracellular domain of CD38. In the present study, we investigated a possible signal transduction mediated through CD38 in the retinoic acid-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (mAbs). The addition of selected anti-CD38 mAbs to the cells induced rapid tyrosine phosphorylation of the cellular proteins with the molecular weights of 120,000, 87,000 and 77,000; the phosphorylated 120-kDa protein was identified as the c-cbl proto-oncogene product, p120c-cbl. Furthermore, the phosphorylated p 120c-cbl associated with the 85-kDa subunit of phosphatidylinositol 3-kinase. To determine the relationship between the amino acid sequence responsible for the NADase activity and epitopes recognized by the stimulatory mAbs, we produced its carboxy-terminal deletion mutants in COS-7 cells. The mutants with less than 15 amino acids deleted from the carboxyl terminus of the 300-amino acid wild-type molecule still maintained NADase activity, but those with more than 27 amino acids deleted did not. Introduction of site-directed mutation of a cysteine residue (Cys275), located in the 273-285 sequence, completely abolished the NADase activity. These CD38 mutants were also used for an epitope mapping of anti-CD38 mAbs. All the epitopes recognized by the mAbs inducing the tyrosine phosphorylation were mapped on the same Cys275-containing sequence of 273-285. Thus, the discrete carboxy-terminal sequence not only plays a key role in its ecto-NADase activity, but also contains the epitopes of the agonistic anti-CD38 mAbs for the transmembrane signaling. We also found that the agonistic mAbs markedly potentiate superoxide generation induced by the stimulation of G protein-coupled chemotactic receptors. Our results suggested that the stimulation of CD38 might generate an accessory signal(s) to enhance the G protein-mediated signaling, probably though the protein-tyrosine phosphorylation.

ADP-ribosyl Cyclase↗
PubMed1997

Negative growth regulation of oncogene-transformed mammary epithelial cells by tumor inhibitors.

Deregulated expression of Ras and myc oncogenes confers neoplastic transformation in non-tumorigenic mammary epithelial cells. Down-regulation of perturbed biomarkers prior to tumorigenesis may provide a means of effective chemoprevention. In vitro experiments were designed to i) identify specific molecular, endocrine and cellular biomarkers as quantitative end points for preneoplastic transformation, and ii) utilize these end points to evaluate chemopreventive efficacy of selected naturally-occurring and synthetic tumor inhibitors. Stable Ras and myc transfectants exhibited persistent expression of oncogene specific mRNA transcripts, altered estradiol biotransformation and enhanced anchorage-independent growth in vitro prior to tumorigenesis in vivo. Treatment of the transfectants with omega-3-fatty acid, indole-3-carbinol and tamoxifen individually suppressed the perturbed molecular, endocrine and cellular biomarkers in vitro. Thus, suppressed oncogene expression and altered estrogen metabolism may be important determinants for antiproliferative mechanisms in mammary tumor inhibition, providing useful end points for chemopreventive intervention of preneoplasia.

Animals↗
PubMed1997

A small glucagonoma of the pancreas with evident ductular and tubular structures.

A 54-year-old woman was admitted to our department for assessment of a tumor of the pancreas found incidentally on abdominal ultrasonography. Examination revealed a hypovascular 1-cm sized tumor in the body of the pancreas. Surgical examination revealed that the tumor was solitary and located in the pancreas body, with no invasion to the adjacent organs iof lymph node involvement. Distal pancreatectomy, preserving the spleen, was performed. Histologically, the tumor was a glucagonoma with evident ductular and tubular structures, suggesting that its site of origin was ductal epithelia.

Female↗
PubMed1997

Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells.

The present study was performed to elucidate the mechanism underlying the anti-atherogenic action of estrogen. We investigated the effect of estrogen on intimal thickening of the rat femoral artery induced by cuff placement and further examined the effect of estrogen on migration and proliferation of vascular smooth muscle cells (VSMCs) in culture. Intimal thickening was significantly greater in males than in control females. Intimal thickening in females was increased to the level in males by ovariectomy. Estrogen replacement to ovariectomized rats reversed this effect. Proliferating cell nuclear antigen immunohistochemistry showed that in vivo proliferation of VSMCs contributed to the difference in intimal thickening. There was no difference in blood pressure and serum lipids, suggesting that estrogen directly acted on artery and inhibited intimal thickening. 17 beta-Estradiol (E2, 1-100 nmol/l) inhibited migration of cultured rat VSMCs, assayed using a microchemotaxis chamber, in a concentration-dependent manner. E2 (0.01-100 nmol/l), but not progesterone or testosterone, also inhibited [3H]thymidine incorporation in rat VSMCs in a concentration-dependent manner. Indomethacin, NG-monomethyl-L-arginine and methylene blue did not influence the inhibitory action of E2 on [3H]thymidine incorporation, suggesting that prostanoids and nitric oxide are not involved in the action of E2. E2 did not provoke VSMC injury, as measured by the release of incorporated [3H]2-deoxy-D-glucose. These results suggest that the inhibition of migration and proliferation of VSMCs contributes to the inhibitory effect of estrogen on intimal thickening.

Animals↗
PubMed1997

In vitro confirmation of newly established lung cancer cell lines using flow cytometry and multicellular tumor spheroids.

We report on a simplified method of cytomorphological in vitro confirmation of newly established lung cancer cell lines by using multicellular tumor spheroids (MTS) and flow cytometry (FCM). Eleven cell lines were established from 11 patients with lung cancer. The MTS were produced by culturing cells in agar-coated dish. Cytomorphological studies were made using smears of crushed MTS and frozen sections of MTS. The MTS were fixed doubly with paraformaldehyde and osmic acid for scanning and transmission electron microscopy. Bivariate fluorescence of fluorescein isothyocyanate (FITC, tumor associated antigen, TAA) and propidium iodide (DNA) were measured by FCM. The MTS grew anchorage-independently. Cytopathological and electron microscopic findings of MTS were similar to those of the original clinical specimens. The DNA index and TAA were useful in evaluating the presence or absence of contamination by cells of non-tumor origin. The new cell lines satisfied a minimum of four conditions to confirm their establishment: (a) they originated from humans, (b) they were cytomorphologically identified with specimens from primary lesions, (c) they showed tumorigenicity, and (d) they were free from contamination by cells of different origin. From these findings, the establishment of new cell lines can be confirmed in vitro by using MTS and FCM.

Antigens, Neoplasm↗