Biomedical subjects

M Sawada

Publications and source records attributed to M Sawada.

At least 19 recordsLinked to original sources

PubMed1992

The neuropeptide, neuromedin C, activates a potassium current in mouse macrophages.

Neuromedin C (NmC) induced an outward current (Io(NmC)) in macrophages. Reversal potentials were dependent on external K+ concentrations ([K+]o) and independent of [Cl-]o. Tetraethylammonium (TEA) and quinidine effectively suppressed Io(NmC). Charybdotoxin (ChTX) and apamin had little effect. Io(NmC) was abolished in Ca(2+)-free EGTA-containing solution. These results suggest that MnC activates a Ca(2+)-dependent K+ current (IK,Ca) and can modulate activities in macrophages.

Amino Acid Sequence↗

PubMed1992

Down regulation of CD4 expression in cultured microglia by immunosuppressants and lipopolysaccharide.

Microglia, brain macrophages, are thought to be the primary target of HIV-1 infection in the brain, because they exclusively express the CD4 antigen which is effectively used for viral entry. The expression of CD4 mRNA in cultured microglia could be detected by the reverse-PCR method. Using this and immunohistochemical staining, we found that the immunosuppressants cyclosporin A and FK506 decreased CD4 expression in cultured murine microglia without causing any significant decrease in cell viability. FK506 was more potent than cyclosporin A. Lipopolysaccharide also decreased CD4 mRNA expression in microglia. The effects of immunosuppressants and lipopolysaccharide seemed to be specific for microglia since these chemicals did not alter the CD4 expression in lymphocytes or peritoneal macrophages. These agents, if modified to pass through the blood-brain barrier, may prevent viral spread of HIV-1 infection in the central nervous system and the AIDS-dementia complex.

Animals↗

PubMed1992

Expression of VLA-4 on thymocytes. Maturation stage-associated transition and its correlation with their capacity to adhere to thymic stromal cells.

The present study investigates the expression of VLA-4 on thymocytes at various stages of maturation and their capacity to adhere to thymic stromal cells. Whole thymocytes were stained with anti-CD4 and anti-CD8, as well as anti-VLA-4 antibodies. Flow microfluorometric analyses revealed that a) most of CD4-8- (double negative DN) and CD4-8intermediate thymocyte populations expressed large amounts of VLA-4, b) the levels of VLA-4 were considerably and markedly reduced on CD4+8+ (double positive DP) and single positive (SP) (CD4+8- or CD4-8+) populations, respectively. This contrasted with an increase in the levels of LFA-1 along with thymocyte maturation. DN, DP, and SP subsets were isolated and examined for their capacity to express VLA-4 and to adhere to fibronectin (FN) molecules as well as thymic stromal cells expressing FN. DN, DP, and SP subsets were confirmed to express the respective high, low, and very low levels of VLA-4, respectively. Approximately 70% of DN thymocytes became bound to FN-precoated culture plates, whereas 30 to 40% of DP and only 10 to 20% of SP cells adhered to FN. Similar patterns of adhesion were observed between these thymocyte subsets and thymic stromal monolayers. The binding of the DN subset to FN-plates or thymic stromal monolayers was inhibited only marginally by the RGDS peptide, but was efficiently inhibited by V10 peptide (cell-binding sequence that is located in the V region on FN and reacts with the VLA-4 integrin) or anti-VLA-4 antibody. Anti-VLA-4 antibody plus RGDS peptide strongly inhibited DN cell binding to FN-coated plates and thymic stromal monolayers. These results indicate that i) VLA-4 expressed on DN thymocytes functions as an important integrin for interacting with thymic stromal cells; ii) the expression level of this integrin decreases with the progress of thymocyte maturation, and iii) most of the mature thymocytes (SP) are rendered less adhesive to thymic stromal cells by reducing the level of VLA-4 expression.

Animals↗

PubMed1992

Induction of cytokines in glial cells by trans activator of human T-cell lymphotropic virus type I.

Human T-cell lymphotropic virus type I (HTLV-I), the cause of adult T-cell leukemia, is also associated with the neurological disease, TSP/HAM (tropical spastic paraparesis/HTLV-I associated myelopathy). The HTLV-I genome encodes a protein, Tax, that trans activates viral and cellular gene transcription. To understand the mechanisms for the production of cytokines by HTLV-I in nervous tissue, we examined their expression in glial cells which carried the Tax-expressing vector. We demonstrated that Tax expression enhanced the production by glial cells of interleukin (IL)-1, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF) beta. We suggest that the excessive production of cytokines in nervous tissue may play a role in pathogenesis of TSP/HAM. Glial cells that carry the tax gene may provide a model useful for in vitro study of the mechanism of production of cytokines in the nervous system.

Animals↗

PubMed1992

Induction of two K+ currents by complement component C5a in mouse macrophages.

Puff application of complement component C5a (5 x 10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced two kinds of outward current at a holding potential of -68 mV, a slowly-rising sustained outward current and a spike-like transient outward current. Quinidine (2 x 10(-4) M) and tetraethylammonium (10(-2) M) partially suppressed both types of outward current. Charybdotoxin (2 x 10(-6) M) markedly suppressed the spike-like outward current. Reversal potentials in bath solutions of different external K+ concentrations were dependent only on K+ concentrations. The transient current was not suppressed in Ca(2+)-free EGTA-containing solution, but was completely abolished in BAPTA-containing solution. One kind of single channel responding to C5a, which has a single-channel conductance of 29 pS, was recorded from cell-attached patches. These results suggest that C5a activates a Ca(2+)-dependent and another type of K+ current.

Animals↗

PubMed1992

Establishment and characterization of human uterine leiomyosarcoma heterotransplanted into nude mice.

Human uterine leiomyosarcoma is a rare gynecological malignancy with a generally poor prognosis. We have established a human uterine leiomyosarcoma tumor line in nude mice, designated UTS-1, and describe the characteristics of this tumor. The UTS-1 tumor doubled in 12.1 days and retained the histological characteristics of leiomyosarcoma, even after 14 serial generations. Ultrastructurally, the tumor is characterized by nuclear pleomorphism typical of smooth muscle, intracytoplasmic filaments with dense bodies, a relative paucity of micropinocytotic vesicles, and an incomplete external lamina. Immunohistochemically, the UTS-1 cells reacted with antibodies against vimentin, desmin, smooth-muscle actin and myosin, but not with antibodies against keratin, CEA and S-100 protein. Serum levels of AFP, CA125, CEA and SCC ranged within normal limits in tumor-bearing mice. The serum level of immunosuppressive acidic protein correlated well with an activity of the tumor. Estrogen and progesterone receptors were not detected in the tumor. Chromosomal analysis showed a human karyotype with some marker chromosomes and a modal number of 85 chromosomes. The UTS-1 tumor should prove a useful model to explore the biological characteristics and treatment of human uterine leiomyosarcoma.

Animals↗

PubMed1992

TNF alpha induces IL-6 production by astrocytes but not by microglia.

Astrocytes and microglia, both produced interleukin-6 (IL-6) in culture by lipopolysaccharide (LPS) stimulation. IL-6 activity was detected 3-5 h after LPS stimulation and reached a maximum at 10 h. Microglia responded faster than astrocytes. Tumor necrosis factor alpha and interleukin 1 also induced IL-6 mRNA and biological activity in astrocytes, but not in microglia. Among these stimuli, LPS was the most potent inducer of IL-6 production by astrocytes. Our results suggest that different regulatory mechanisms for cytokine production exist in glial cells. The possible roles of astrocytes and microglia in CNS immune responses are also discussed.

Animals↗

PubMed1992

Activation of K+ current in macrophages by platelet activating factor.

Puff application of platelet activating factor (10(-8) M) onto peritoneal macrophages from thioglycollate-stimulated mice induced an outward current at a holding potential of -63 mV. The current was suppressed by an antagonist Y-24180 but not by CV-3988. Charybdotoxin (10(-6) M) suppressed the current. Reversal potentials were dependent on external K+ concentrations. The current was not suppressed in Ca(2+)-free EGTA-containing solution but was completely abolished in BAPTA-AM containing solution. These results suggest that platelet activating factor activates a Ca(2+)-dependent K+ channel.

Animals↗

PubMed1992

Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat.

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro-elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca(2+)-sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme-mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.

Aging↗

PubMed1992

Interleukin-2 inhibits the GABA-induced Cl- current in identified Aplysia neurons.

The effects of extracellularly applied recombinant human interleukin-2 (rhIL-2) on the gamma-aminobutyric acid (GABA)-induced Cl- current recorded from identified neurons (R9 and R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied rhIL-2 (10-40 U/ml) reduced the GABA-induced current in the neurons without affecting resting membrane conductance and the holding current. The suppressing effect of rhIL-2 on the current was completely reversible. Heat-inactivated rhIL-2 was without effect. These results suggest that the immunomodulator IL-2 can modulate the GABA-induced response in the nervous system.

Animals↗

PubMed1992

Reduction of the acetylcholine-induced K+ current in identified Aplysia neurons by human interleukin-1 and interleukin-2.

1. Effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodai were investigated with voltage-clamp and pressure ejection techniques. 2. Bath-applied rhIL-1 and rhIL-2 (10-40 U/ml) reduced the ACh-induced current in the neurons without affecting the resting membrane conductance and holding current. 3. The suppressing effects of these cytokines on the current were completely reversible. 4. Heat-inactivated rhIL-1 and rhIL-2 were without effect. 5. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the ACh-induced response in the nervous system.

Acetylcholine↗

PubMed1992

A prostacyclin analogue reduces free radical generation in heart-lung transplantation.

The mechanism by which prostacyclin acts to prevent in vivo reperfusion injury is still uncertain. This study was therefore undertaken to assess the effect of a stable prostacyclin analogue (OP 41483-alpha-CD [OP]) on oxygen-derived free radicals after heart-lung transplantation. OP was administered to the heart-lung graft through the pulmonary artery for 25 minutes encompassing the reperfusion process. Free radicals were directly measured by electron spin resonance spectroscopy. The radical intensities of pulmonary venous blood were significantly lower in the OP group than in the control group, suggesting that fewer free radicals were generated in the lungs of the OP group. The cardiac and respiratory function were better in the OP group than in the control group. The lung is the primary source of oxygen free radical attack, and the beneficial action of OP on free radical generation is almost exclusively restricted to the lung and does not apply to the heart. This result suggested that OP probably is effective in inhibiting free radical generation from the endothelium.

Adenosine Diphosphate↗

PubMed1992

Stable expression of cytochrome P450IIIA7 cDNA in human breast cancer cell line MCF-7 and its application to cytotoxicity testing.

A mammalian cell expression plasmid containing cytochrome P450IIIA7 complementary DNA was constructed. Breast cancer cells (MCF-7) were transfected with the plasmid and neomycin-resistant selection marker plasmid. We established three cell lines, termed M13, M21, and M27, which expressed the cytochrome P450IIIA7 as examined by RNA blot and immunoblot analyses. These cell lines showed 8- to 10-fold higher sensitivity against aflatoxin B1 compared to parental MCF-7 cells, suggesting that cytochromes P450IIIA7 expressed in the cells were responsible for the production of the cytotoxic metabolite of aflatoxin B1.

Adult↗

PubMed1992

Ferric(III) ions inhibits copper(II)/hydrogen peroxide-catalyzing lipid peroxidation in human erythrocyte membranes.

1. Effect of ferric ions (Fe3+) on the lipid peroxidation catalyzed by copper ions (Cu2+) and hydrogen peroxide (H2O2) was studied in human erythrocyte membranes. 2. The formation of thiobarbituric acid-reactive products elicited by CuCl2/H2O2 was inhibited by FeCl3 in a concentration-dependent manner; 0.25 mM FeCl3 were enough to cause 50% inhibition of the formation of peroxides. 3. The inhibitory effect of FeCl3 is not due to competition against Cu2+. 4. FeCl3 inhibited the initiation, but did not inhibit the propagation of Cu2+/H2O2-catalyzing lipid peroxidation. 5. In the heat- or trypsin-treated erythrocyte membranes, FeCl3 had no inhibitory effect on Cu2+/H2O2-catalyzing lipid peroxidation. 6. Sodium azide, an inhibitor of catalase, had no effect on the inhibitory effect of FeCl3. 7. These results suggest that a protein factor(s), which is not catalase, is involved in the inhibition of Cu2+/H2O2-catalyzing lipid peroxidation by Fe3+.

Copper↗

PubMed1992

Stable expression of monkey cytochrome P-450IA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B1.

A monkey cytochrome P-450IA1 cDNA (MKah1) was transfected into Chinese hamster CHL cells using a vector containing the SR alpha promoter, and sublines stably expressing P-450IA1 were established. The cells showed 25-fold higher sensitivity to the cytotoxic effect of aflatoxin B1 than the parental CHL cells. This hypersensitivity was almost completely suppressed by alpha-naphthoflavone, which is a known specific inhibitor of P-450IA. The cells expressing P-450IA1, but not CHL cells, showed a positive response to aflatoxin B1 in an assay for mutagenicity at the HGPRT locus.

Aflatoxin B1↗

PubMed1992

A PCR method for the quantitative assessment of mRNA for laminin A, B1, and B2 chains.

Laminin, a basement membrane glycoprotein, is involved in the development of normal kidney and its dysregulation contributes to glomerulosclerosis in renal disease. Studies designed to assess the regulation of this molecule at the level of transcription have been hindered by the relatively low abundance of the mRNA, making standard techniques such as Northern hybridization and RNase protection difficult and inaccurate. In this report, we have utilized the polymerase chain reaction (PCR) to quantitate differences in laminin mRNA expression during normal development of the mouse kidney. We have constructed a synthetic template to be used as an internal standard for mRNA quantitative of laminin chains A, B1 and B2, and beta-actin. This DNA template can be used to generate complementary RNA which can be reverse transcribed and amplified simultaneously with 0.5 microgram of total cellular mRNA allowing for accurate and absolute quantitation of laminin mRNA by PCR.

Animals↗

PubMed1992

Stimulation of phospholipase A2 by xanthine oxidase in the rat corpus luteum.

The ability of the superoxide radical (SOR) generated by xanthine oxidase to activate phospholipase A2 (PLA2) was examined in microsomes prepared from luteinized rat ovaries. Treatment of microsomes with xanthine oxidase resulted in a rapid burst in SOR formation followed by an increase in PLA2 activity. Stimulation of PLA2 activity was dose related and similar in microsomes prepared from control or prostaglandin F2 alpha (PGF2 alpha)-treated rats. Activation was inhibited by the antioxidants, vitamin E and nordihydroguaiaretic acid, and by superoxide dismutase and catalase, which metabolize SOR and H2O2 to remove reactive oxygen species from the cell. The stimulation of PLA2 activity by xanthine oxidase was dependent upon the addition of calcium ions, and it was highest in samples in which cytosol was added to membranes. These results indicate that the SOR and/or H2O2 may mediate PLA2 activation, which may be involved in the luteolytic process.

Acetophenones↗

PubMed1992

Establishment of mouse oligodendrocyte/type-2 astrocyte lineage cell line by transfection with origin-defective simian virus 40 DNA.

A permanent glial cell line has been established from the neonatal mouse primary mixed glial cell cultures by transfection with replication origin-defective simian virus 40 DNA. This cell line, designated OS3, has morphological similarity to type-2 astrocyte and expresses an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), when cultured in the presence of 10% calf serum (CS). OS3 cells do not express the O4 antigen, galactocerebroside (GalC) and A2B5 under this culture condition. When cultured in a medium containing 2% CS or a chemically defined medium, these cells undergo morphological transformation. Some of these cells express O4 antigen and/or GalC, and the percentage of GFAP positive cells decreases under these conditions. Thus depending on the culture conditions, the OS3 cells display either type-2 astrocyte properties or immature oligodendrocyte characteristics. Furthermore, the OS3 cells show similar responses to the various growth factors as do oligodendrocyte/type-2 astrocyte (O-2A) progenitors. Therefore, the OS3 cell line is an unique mouse bipotential permanent O-2A lineage cell line which may be useful to analyze the developmental properties of these glial cells.

Animals↗