Biomedical subjects

S Nagata

Publications and source records attributed to S Nagata.

At least 163 records · Page 9Linked to original sources

PubMed1998

Simultaneous determination of bile acids in rat liver tissue by high-performance liquid chromatography.

A method for the simultaneous determination of bile acids in rat liver tissue by high-performance liquid chromatography was developed. Without prior fractionation and alkaline hydrolysis, 30 unconjugated, glycine- and taurine-conjugated bile acids were detected by post-column enzymatic reaction and fluorescence detection. They were separated on a reversed-phase column using a linear gradient solvent system of 10 mM tribasic ammonium phosphate-acetonitrile-methanol (44:12:5, v/v/v) and 20 mM dibasic ammonium phosphate-acetonitrile-methanol (2:1:2, v/v/v). The limits of detection were 1-5 pmol, and calibration curves were linear for concentrations ranging between 10 and 4000 pmol per 10 microl injection. This rapid and reliable method is effective for measuring bile acid levels in liver tissue not only of rats but also of patients with hepatobiliary and other diseases.

Animals↗

PubMed1998

Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution.

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.

Animals↗

PubMed1998

Nucleotide sequence, cloning, and overexpression of the D-threonine dehydrogenase gene from Pseudomonas cruciviae.

D-Threonine dehydrogenase (EC 1.1.1) catalyses the oxidation of the 3-hydroxyl group of D-threonine. The nucleotide sequence of the structural gene, dtdS, for this enzyme from Pseudomonas cruciviae IFO 12047 was determined. The dtdS gene encodes a 292 amino acid polypeptide. The enzyme was overproduced in Escherichia coli cells; the activity was found in cell extracts of the clone. The enzyme showed high sequence similarity to 3-hydroxyisobutyrate dehydrogenases. This is the first example showing the primary structure of an enzyme catalysing the NADP(+)-dependent dehydrogenation of D-threo-3-hydroxyamino acids.

Alcohol Oxidoreductases↗

PubMed1998

Molecular cloning and characterization of human caspase-activated DNase.

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.

Amino Acid Sequence↗

PubMed1998

Fas/FasL interaction is not involved in apoptosis of activated CD4+ T cells upon HIV-1 infection in vitro.

In HIV-1-infected individuals, Fas expression and Fas/FasL-mediated apoptosis of mature T cells are known to increase compared with those in normal individuals. To elucidate a relation between acute HIV-1 infection and the regulation of Fas/FasL system upon T-cell activation, resting CD4+ T cells were acutely infected or uninfected with HIV-1 and subsequently activated by phorbol myristate acetate and ionomycin (PMA/IM). Four days after infection, when HIV-1 env gp120 is expressed in more than one half of activated T cells, Fas/FasL expression was analyzed by flow cytometry, and apoptosis-inducing activity of these activated primary CD4+ T cells on Fas+ Jurkat cells was examined. The level of Fas or FasL expression was not altered during acute HIV-1 infection. The enhanced apoptosis-inducing activity upon HIV-1 infection was observed in some individuals, but its activity was not Fas/FasL-mediated. These results indicate that HIV-1 infection is not necessarily associated with either upregulation of Fas/FasL expression or Fas/FasL-mediated apoptosis.

Antibodies, Monoclonal↗

PubMed1998

Levels of soluble Fas ligand in myocarditis.

Serum levels of soluble Fas ligand (sFasL) increased with the severity of congestive heart failure (p <0.01), and the percentages of apoptotic myocytes detected by in situ DNA nick-end labeling were significantly higher in the patients with increased levels of sFasL than in those with normal levels of sFasL (p <0.05). These findings indicated that sFasL may play an important role in pathogenesis of myocarditis.

Adult↗

PubMed1998

Specific detection of phosphatidylinositol 3,4,5-trisphosphate binding proteins by the PIP3 analogue beads: an application for rapid purification of the PIP3 binding proteins.

Phosphatidylinositol (PI) 3-kinase is known as one of the key molecules involved in the various biological events such as vesicle trafficking, cytoskeletal rearrangements and cell survival. T clarify the molecular basis underlying these events, we have tried to identify the proteins that can interact with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid product of PI3-kinase. Using a new PIP3 analogue, PIP3-APB, we synthesized an affinity column for PIP3 binding proteins. This enabled us to purify and identify several PIP3 binding proteins such as Tec tyrosine kinase, Gap1m, and Akt, as the candidates for the downstream molecules of PI3-kinase. All of these proteins contain PH domains, possible binding sites for phospholipids. Studies with various deletion mutants of Tec or Gap1m revealed that their PH domains are indeed the binding sites for PIP3. These results demonstrate that this PIP3-analogue binds various PIP3 binding proteins with high specificity and may be useful to elucidate the downstream mechanisms of PI3-kinases-mediated signaling pathways.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Elevation of the serum Fas ligand in patients with hemophagocytic syndrome and Diamond-Blackfan anemia.

Fas ligand (FasL) is a membrane protein that is expressed in activated T cells and natural killer cells. FasL binds to Fas on target cells and induces apoptosis. There exists a soluble form of FasL (sFasL), and sFasL also induces apoptosis of Fas-bearing cells. The serum sFasL concentrations were reported to be elevated in patients with large granular lymphocytic leukemia and natural killer cell lymphoma. In this study, we have measured serum sFasL concentrations in other hematological disorders, including severe aplastic anemia (SAA), hemophagocytic lymphohistiocytosis (HLH), and Diamond-Blackfan anemia (DBA). The serum sFasL concentration of age-matched healthy controls was 0.16 +/- 0.11 ng/mL (mean +/- SD, n = 22). The serum sFasL levels in the patients with HLH and DBA were 3.75 +/- 3.82 (n = 19; P < .0001, HLH v control) and 2.76 +/- 2.43 ng/mL (n = 6; P = .012, DBA v control), respectively. Serum interferon-gamma concentration was elevated in the patients with HLH (1.61 +/- 2.62 ng/mL) but not in those with DBA (below the detectable level). These results suggest that the Fas-FasL system plays a role, at least in part, in the pathophysiology of HLH and DBA.

Adolescent↗

PubMed1998

Transgenic expression of Fas in T cells blocks lymphoproliferation but not autoimmune disease in MRL-lpr mice.

Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1+ B220+), but still accumulated B cells (Thy-1- B220+); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells.

Animals↗

PubMed1998

Myeloid-specific transcriptional activation by murine myeloid zinc-finger protein 2.

Myeloid zinc finger protein 2 (MZF-2) is a zinc-finger transcription factor that is expressed in myeloid cells, particularly in the cells committed to the neutrophilic lineage. Here we examine the ability of murine MZF-2 (mMZF-2) to activate transcription. The mMZF-2 protein binds to a DNA element (MZF-binding site) through its zinc-finger domain. When the intact mMZF-2 was cotransfected with a reporter gene, it did not activate transcription. However, N-terminal deletion mutants greatly enhanced transcription specifically in myeloid cells. Furthermore, in an in vivo competition assay, the middle region of MZF-2 inhibited the mMZF-2-mediated transcription activation. These results suggest that mMZF-2 is a transcriptional factor that can specifically work in myeloid cells and can be divided into at least three functional domains. The N-terminal domain inhibits transactivation by masking the effect of the activation domain. The middle region recruits a coactivator, which is responsible for myeloid-specific transcriptional activation. The C-terminal zinc-finger domain functions as a DNA-binding domain.

Animals↗

PubMed1998

Abrogation of Fas-induced fulminant hepatic failure in mice by hepatocyte growth factor.

Excessive activity of the Fas system in the liver is an essential event and contributor to fulminant hepatic failure, whose prognosis is extremely poor with high mortality due to lack of effective therapy. Administration of agonistic anti-Fas antibody to mice rapidly led to massive liver apoptosis and fulminant hepatic failure. In contrast, administration of human recombinant hepatocyte growth factor (HGF) abrogated Fas-induced massive liver apoptosis and the lethal hepatic failure. Addition of anti-Fas antibody to hepatocytes in primary culture induced cell death, but Fas-mediated cell death was potently suppressed by HGF. HGF strongly induced Bcl-xL expression and subsequently blocked Fas-mediated signaling pathway upstream of CPP32 in the liver. These results implicate a potential therapeutic usage of HGF for treatment of fulminant hepatic failure.

Animals↗

PubMed1998

Density dependent elevation of phosphatidylinositol-3 kinase level in rat 3Y1 cells.

We have explored the levels of phosphatidylinositol-3 kinase protein during culture of rat 3Y1 cells. Confluent cell cultures exhibited a higher level of phosphatidylinositol-3 kinase compared with that of growing cells. After replating of the cells on fresh dishes, the level of phosphatidylinositol-3 kinase returned to that of growing cells within 24h. This density-dependent regulation of the phosphatidylinositol-3 kinase level was not lined to cell growth, because growth arrest by serum starvation did not cause elevation of the phosphatidylinositol-3 kinase level. Northern blotting analysis revealed that this regulation was based on the transcriptional level. After cell growth was arrested by contact inhibition, elevation of the level of phosphatidylinositol-3,4,5-trisphosphate was detected suggesting that phosphatidylinositol-3 kinase was activated in these cells. These effects were not seen in src-transformed 3Y1 cells, suggesting that this regulation was lost in transformed cells.

Animals↗

PubMed1998

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD.

The homeostasis of animals is regulated not only by the growth and differentiation of cells, but also by cell death through a process known as apoptosis. Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA. A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic fraction of mouse lymphoma cells. CAD is a protein of 343 amino acids which carries a nuclear-localization signal; ICAD exists in a long and a short form. Recombinant ICAD specifically inhibits CAD-induced degradation of nuclear DNA and its DNase activity. When CAD is expressed with ICAD in COS cells or in a cell-free system, CAD is produced as a complex with ICAD: treatment with caspase 3 releases the DNase activity which causes DNA fragmentation in nuclei. ICAD therefore seems to function as a chaperone for CAD during its synthesis, remaining complexed with CAD to inhibit its DNase activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing CAD to enter the nucleus and degrade chromosomal DNA.

Amino Acid Sequence↗

PubMed1998

Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis.

Various molecules such as cytokines and anticancer drugs, as well as factor deprivation, rapidly induce apoptosis (programmed cell death), which is morphologically characterized by cell shrinkage and the blebbing of plasma membranes and by nuclear condensation. Caspases, particularly caspase 3, are proteases that are activated during apoptosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin. Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA. In the accompanying Article, we have identified and molecularly cloned a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD). Here we show that caspase 3 cleaves ICAD and inactivates its CAD-inhibitory effect. We identified two caspase-3 cleavage sites in ICAD by site-directed mutagenesis. When human Jurkat cells were transformed with ICAD-expressing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation. The ICAD transformants were also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase. Our results indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, and that ICAD works as an inhibitor of this process.

Animals↗

PubMed1998

Natural killer cell-derived large granular lymphocyte lymphoma of lung developed in a patient with hypersensitivity to mosquito bites and reactivated Epstein-Barr virus infection.

A 17-year-old female developed natural killer (NK) cell-derived large granular lymphocyte (LGL) lymphoma of the lung. She had a past history of hypersensitivity to mosquito bites (HMB). After an eight-year chronic, active Epstein-Barr virus (EBV) infection, she developed multiple lung lesions and pleural effusion. In the effusion, 60% of the cells were LGL. They were CD2+, 3-, 16+, 56+, 57+, 45RO+/RA + weak, and possessed strong NK activity. No rearrangement of T-cell-receptor genes was detected. From all these results, a diagnosis of NK-LGL lymphoma of the lung was made. EB virus DNA was detected in cells infiltrating the pleural effusion. The clonality of the LGLs was determined by Southern blot hybridization with the terminal repeat sequence of EB virus as a probe, and by chromosomal abnormalities. The patient died from respiratory failure. Necropsy of the lung revealed diffuse lymphoma composed of polymorphic cells with typical angiocentric lesions. Reportedly, lymphomas of NK lineage show predominantly extranodal involvement, and primary lung lesions are rare. In the pleural effusion of the present case, abnormally high levels of soluble Fas ligand, interleukin-10 and interferon gamma were detected. This hypercytokinemia, reflecting the microenvironment of lymphoma cells, may play a role in the progression of the lymphoma and organ injury in the lung.

Adolescent↗

PubMed1998

Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE: To investigate the expression and function of Fas ligand (FasL),which can be in a membrane-bound or soluble form, in the joints of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The concentration of soluble FasL (sFasL) in serum and synovial fluid (SF) from 24 OA and 38 RA patients was measured using an enzyme-linked immunosorbent assay. The expression of FasL on SF lymphocytes (SFL) and peripheral blood lymphocytes (PBL) was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. A cytotoxic killing assay of membrane-bound FasL and purified sFasL against cultured synovial cells was also performed. RESULTS: Soluble FasL was detected in the SF of patients with RA and OA, but not in their serum. The concentration of SF sFasL was remarkably higher in patients with severe RA than in patients with mild RA or with OA. RT-PCR showed that SFL, but not PBL, from RA patients expressed messenger RNA for FasL. Membrane-bound FasL induced apoptosis in cultured synovial cells from the RA and OA patients, but naturally processed human sFasL did not. CONCLUSION: SFL from RA patients expressed FasL, and cleaved sFasL accumulated in the SF of inflamed joints. The different killing activity of membrane-bound FasL and sFasL against synovial cells may regulate Fas-mediated apoptosis in synovial cells.

Aged↗

PubMed1998

Myocardial glucose metabolism in patients with hypertrophic cardiomyopathy: assessment by F-18-FDG PET study.

In an investigation of myocardial metabolic abnormalities in hypertrophic myocardium, the myocardial glucose metabolism was evaluated with F-18-fluorodeoxyglucose (FDG) positron emission tomography (PET) in 32 patients with hypertrophic cardiomyopathy, and the results were compared with those in 9 patients with hypertensive heart disease. F-18-FDG PET study was performed in the fasting and glucose-loading states. The myocardial regional %dose uptake was calculated quantitatively. The average regional %dose uptake in the fasting state in the patients with asymmetric septal hypertrophy and dilated-phase hypertrophic cardiomyopathy was significantly higher than that in the patients with hypertensive heart disease (0.75 +/- 0.34%, 0.65 +/- 0.25%, and 0.43 +/- 0.22%/100 g myocardium, respectively). In contrast, the average %dose uptake in the glucose-loading state in the patients with asymmetric septal hypertrophy and dilated-phase hypertrophic cardiomyopathy was not significantly different from that in patients with hypertensive heart disease (1.17 +/- 0.49%, 0.80 +/- 0.44% and 0.99 +/- 0.45%, respectively). The patients with apical hypertrophy had also low %dose uptake in the fasting state (0.38 +/- 0.21%) as in the hypertensive heart disease patients, so that the characteristics of asymmetric septal hypertrophy and dilated-phase hypertrophic cardiomyopathy are considered to be high FDG uptake throughout the myocardium in the fasting state. Patients with apical hypertrophy are considered to belong to other disease categories metabolically. F-18-FDG PET study is useful in the evaluation of the pathophysiologic diagnosis of patients with hypertrophic cardiomyopathy.

Blood Glucose↗

PubMed1998

Induction of blood cells in Xenopus embryo explants.

A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap. XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants. In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes. At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized. Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia. In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant. Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells. Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes. These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis.

Activins↗