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Biomedical subjects

M Sawada

Publications and source records attributed to M Sawada.

At least 253 records · Page 14Linked to original sources
PubMed1994

cDNA cloning of a novel protein tyrosine phosphatase with homology to cytoskeletal protein 4.1 and its expression in T-lineage cells.

Reversible tyrosine phosphorylation plays important regulatory roles in various cellular events including the differentiation and function of lymphocytes. Here we report the cDNA cloning of a non-receptor type protein tyrosine phosphatase, PTP36, which is expressed in murine thymus. PTP36 was a new member of a tyrosine phosphatase subfamily defined by MEG-01 and PTPH1, which had a C-terminal phosphatase domain as well as an N-terminal domain with homology to cytoskeletal-associated proteins like band 4.1, ezrin, and talin. In addition, we found a putative SH3-binding motif in PTP36 but not in MEG-01 or PTPH1. PTP36 was expressed in cells of both hematopoietic and non-hematopoietic origins. In thymocytes subpopulations, PTP36 was preferentially expressed in double positive stage cells. The change of PTP36 expression level along with T cell maturation suggests its involvement in the regulation of T cell development.

Amino Acid Sequence↗
PubMed1994

Clonal analysis of human gynecologic cancers by means of the polymerase chain reaction.

Clonality of human gynecologic cancers was analyzed in small DNA samples prepared from cryostat sections, by means of the polymerase chain reaction (PCR). The method used for clonal analysis was based on restriction fragment length polymorphism of the X-chromosome-linked phosphoglycerokinase (PGK) gene and on the differential methylation of the PGK gene due to random inactivation of 1 of 2 X-chromosomes by methylation in females. Among 52 gynecologic cancers tested, 25 were found to be heterozygous for the BstXI polymorphism of the PGK gene. All the 25 gynecologic cancers (4 cervix, 11 endometrium, 7 ovary and 3 fallopian tube) analyzed by the PCR-based method were monoclonal in origin while adjacent normal tissues were polyclonal. When DNA samples were prepared from widely separated sites of tumors and/or metastatic lesions, every sample was found to be monoclonal, and the same allele of the PGK gene was inactivated in each case. These results demonstrate that clonal analysis by PCR offers a good method for studying clonality in small DNA samples prepared from cryostat sections of tumors. This method could be applied to distinguish between benign and malignant gynecologic lesions.

Adult↗
PubMed1994

Stable expression of human CYP1A2 and N-acetyltransferases in Chinese hamster CHL cells: mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.

In order to investigate the metabolic activation pathway of food-derived heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), cultured cell lines which stably expressed human cytochrome P4501A2 (CYP1A2) and N-acetyltransferases (NATs) were developed by the method of complementary DNA (cDNA) transfection. First, a cell line expressing CYP1A2, designated A2R-5, was established from the cell line CR-68, which was previously established by introducing NADPH-cytochrome P-450 reductase cDNA into Chinese hamster CHL cells. The expression of CYP1A2 in the transfected cells was confirmed by determining sensitivity to aflatoxin B1. As the next step, the A2R-5 as well as CR-68 cells were further transfected with human monomorphic NAT (NAT1) or polymorphic NAT (NAT2) cDNAs. The expression of NAT in the transfected cells was confirmed using p-aminobenzoic acid and sulfamethazine as substrates, while no activity was seen in parental CR-68 and A2R-5 cells. The cell line, ANP-25, which expressed both CYP1A2 and NAT2, was approximately 370- and 100-fold more sensitive to IQ and MeIQx, respectively, than parental CR-68 cells in cytotoxicity assays. There were no clear differences in sensitivity to both compounds among CR-68, A2R-5, and the cell lines which expressed NAT1 alone, NAT2 alone, and CYP1A2 plus NAT1. Mutagenicity of IQ and MeIQx at the hypoxanthine-guanine phosphoribosyltransferase locus was also detectable only in ANP-25 cells but not in A2R-5 or the cell line expressing CYP1A2 plus NAT1. From these results, it is proposed that both CYP1A2 and NAT2 (but not NAT1) are required for mutagenic activation of these compounds, implying that acetylator polymorphism may be an important risk factor in the carcinogenicity of these compounds.

Acetyltransferases↗
PubMed1994

Effect of dibutyryl cyclic AMP on morphologic features and marker production of human cervical argyrophil small-cell carcinoma cell line.

The effect of dibutyryl cyclic AMP (dB-cAMP) on the morphologic features and marker production of a human cervical argyrophil small-cell carcinoma (ASCC) cell line was examined. Following 1-5 days' exposure to 5 mM dB-cAMP, morphologic differentiation as defined by the expression of cytoplasmic processes (stellate cells) was observed. The number of stellate cells depended on the dose of dB-cAMP and incubation time. Shortly after removal of dB-cAMP from the culture medium, the treated cells returned to their original spherical shape. dB-cAMP caused a reduction in the growth rate of cells which recovered after removal of the agent. The morphological changes appeared not to be the result of growth inhibition by dB-cAMP, because the cells maintained in a serum-free medium did not show any change in shape. Electron microscopic study revealed the development of intracytoplasmic microtubules, microfilaments, and an increase in the number of neurosecretory granules in the treated cells. The levels of neuron-specific enolase, serotonin and gastrin in treated cells were significantly higher than those in untreated controls. These findings indicate that a reversible differentiation of cultured ASCC cells into neuroendocrine cells occurs in a growth medium containing dB-cAMP.

Biomarkers, Tumor↗
PubMed1994

A flow cytometric assay reveals an enhancement of phagocytosis by platelet activating factor in murine peritoneal macrophages.

Phagocytosis of fluorescein isothiocyanate-labeled latex beads by peritoneal macrophages from thioglycollate-stimulated mice was examined in vitro by means of flow cytometry. This assay revealed that platelet activating factor (PAF), a known inflammatory mediator, enhanced the phagocytosis in a dose-dependent manner. The enhancement was completely suppressed by a PAF antagonist, Y-24180, and partly suppressed by another antagonist, CV-3988. The phagocytosis of both nonstimulated and PAF-stimulated macrophages was suppressed in Ca(2+)-free ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing solution, but the phagocytosis was enhanced by PAF even in this solution. These results suggest that phagocytosis by macrophages is enhanced by PAF in allergic and inflammatory reactions.

Animals↗
PubMed1994

Inhibition of the calcitonin-induced outward current in identified Aplysia neurons by interleukin-1 and interleukin-2.

1. The effects of bath-applied recombinant human interleukin-1 (rhIL-1) and interleukin-2 (rhIL-2) on the calcitonin (CT)-induced outward current recorded from identified neurons (R9-R12) of Aplysia kurodai were investigated with conventional voltage-clamp and pressure ejection techniques. 2. Micropressure ejection of CT onto the soma of the neuron induced a slow outward current [Io(CT); 4-6 nA in amplitude, 30-40 sec in duration] associated with a decrease in input membrane conductance. 3. Io(CT) was increased by hyperpolarization. 4. The extrapolated reversal potential was +10 mV. Additionally, Io(CT) was sensitive to changes in (Na+)o but not to changes in (K+)o, (Ca2+)o, and (Cl-)o. 5. Micropressure-ejected forskolin produced a slow outward current similar to that induced by CT. 6. Bath-applied rhIL-1 and rhIL-2 (10-40 U/ml) reduced the CT-induced current in identified Aplysia neurons without affecting the resting membrane conductance or the holding current. 7. The inhibitory effects of both cytokines on the current were completely reversible. Heat-inactivated rhIL-1 and rhIL-2 were without effect. 8. These results suggest that the immunomodulators, IL-1 and IL-2, can modulate the CT-induced outward current associated with a decrease in Na+ conductance in the nervous system of Aplysia. Therefore, the study suggests that these cytokines may also serve as neuromodulators.

Animals↗
PubMed1994

Suppression of phagocytosis by adrenocorticotropic hormone in murine peritoneal macrophages.

Phagocytosis of latex beads by peritoneal macrophages was examined by means of flow cytometry (FCM). This assay revealed that adrenocorticotropic hormone (ACTH) suppressed phagocytosis in a dose-dependent manner. ACTH (1-24) was more suppressive than ACTH (1-39). Control phagocytosis was partially suppressed in Ca(2+)-free solution. Phagocytosis was suppressed by ACTH in this solution to the same degree as in the normal solution. Suppression by ACTH was reduced in phosphodiesterase inhibitor-containing solution. These results suggest that (1) ACTH suppresses extracellular Ca(2+)-dependent and -independent phagocytosis, (2) the suppression is not mediated by cAMP and (3) the inhibition of macrophage phagocytosis by ACTH is one of the mechanisms that modulate immune responses in stressful situations.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Induction of MHC class II antigen expression on murine microglia by interleukin-3.

The effects of various cytokines on MHC class II antigen expression were examined in murine microglia. Interleukin-3 (IL-3), as well as interferon-gamma (IFN-gamma), induced MHC class II antigen expression on these cells. IL-3 additionally enhanced MHC class II antigen expression induced by IFN-gamma. The induction of MHC class II antigen expression by IL-3 was not mediated via IFN-gamma production, because the effect was not blocked by antibodies to IFN-gamma. In contrast, granulocyte-macrophage colony-stimulating factor (GM-CSF) did not affect the expression of MHC class II antigen on naive cells and down-regulated IFN-gamma-mediated induction of MHC class II antigen expression on microglia. Because IL-3 and GM-CSF are apparently produced in the central nervous system, MHC class II antigen expression on microglia may be regulated by these cytokines synthesized in the central nervous system.

Animals↗
PubMed1994

Interleukin-4 induces proliferation and activation of microglia but suppresses their induction of class II major histocompatibility complex antigen expression.

We recently found that microglia, brain macrophages, express interleukin-4 (IL-4) receptor mRNA in vitro. Since IL-4 exhibits a variety of functions on the cells of monocyte-macrophage lineage, we examined the effects of IL-4 on the functions of microglia. Recombinant IL-4 induced the proliferation of microglia in a dose- and time-dependent manner as determined by MTT colorimetric assay, [3H]thymidine uptake and bromodeoxyuridine (BrdU) incorporation. IL-4 also synergistically enhanced the proliferation of microglia with such colony-stimulating factors as IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). It also increased acid phosphatase activity and superoxide anion formation by these cells. Despite these positive effects on proliferation and activation, IL-4 suppressed the IFN gamma-induced class II MHC antigen expression in these cells. Since these effects of recombinant IL-4 were inhibited by the addition of monoclonal antibody against IL-4 receptors, the effects of IL-4 on microglia appear to be a specific function via IL-4 receptors. Although microglia and astrocytes produce a variety of immunoregulatory cytokines, neither cell produced IL-4 as determined by bioassay or detection of IL-4 mRNA by RT-PCR method. Thus, the exogenous IL-4 may contribute to the accumulation of microglia in or around inflammatory lesions in the central nervous system, and may be involved in the regulatory mechanisms of microglia.

Animals↗
PubMed1994

Simultaneous determination of human neutrophil elastase inhibitor (ONO-5046) and its metabolite in plasma and urine by direct injection column-switching HPLC.

The direct injection method for the simultaneous determination of a human neutrophil elastase inhibitor (ONO-5046) and its metabolite (ONO-EI-601) in plasma and urine has been developed using a column-switching HPLC system. The system was set up with a pre-treatment column, a pre-concentration column and an analytical column which were connected in series via two automatic switching valves. The calibration lines showed a good linearity for concentrations of ONO-5046 and ONO-EI-601 in a range of 156-20000 ng ml-1 in plasma and 1.56-100 micrograms ml-1 in urine, all correlation coefficients being greater than 0.9999. The limit of quantitation of ONO-5046 was 156 ng ml-1 in plasma, that of ONO-EI-601 was 313 ng ml-1 in plasma, and those of ONO-5046 and ONO-EI-601 were 1.56 micrograms ml-1 in urine. The developed method is rapid and sensitive with automated operation, allowing untreated samples to be analysed every 45 min. The application of the present method to the real plasma and urine samples proved to be useful for pharmacokinetic, toxicological and clinical studies.

Chromatography, High Pressure Liquid↗
PubMed1994

Application of an alpha-sidechain length-specific monoclonal antibody to immunoaffinity purification and enzyme immunoassay of 2,3-dinor-6-keto-prostaglandin F1 alpha from human urine.

2,3-Dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha) is a major urinary metabolite of PGI2 (prostacyclin) and one of the most reliable parameters of PGI2 production in vivo. A mouse was immunized with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha conjugated to bovine serum albumin for preparation of a monoclonal antibody which recognized the difference in the alpha-sidechain length of 6-keto-PGF1 alpha and its 2,3-dinor-metabolite. A sensitive and specific enzyme immunoassay was developed by the solid-phase competition method with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha labeled with peroxidase protein. The detection range of the assay was 14-1200 fmol (IC50 = 120 fmol). The cross-reactivities of the antibody with 6-keto-PGF1 alpha, 6,15-diketo-13,14-dihydro-PGF1 alpha, and other arachidonate metabolites were less than 0.01%. An immunoaffinity column was prepared by coupling the anti-2,3-dinor-6-keto-PGF1 alpha antibody to BrCN-activated Sepharose 4B. Human urine was applied to an octadecylsilyl silica cartridge, and the extract was applied to the immunoaffinity column. This procedure allowed an efficient separation of 2,3-dinor-6-keto-PGF1 alpha from unidentified urinary substances which interfered with immunoassay. Validity of the results obtained by the enzyme immunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

6-Ketoprostaglandin F1 alpha↗
PubMed1994

Determination of plasma tissue factor antigen and its clinical significance.

To investigate the clinical significance of determination of plasma tissue factor (TF) antigen, we have developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for plasma TF, using two different monoclonal antibodies against TF apoprotein, 6B4 (catching antibody) and 5G9 (detecting antibody), and tetramethyl benzidine/H2O2 as substrates. Titration curves of recombinant human TF in buffer containing Triton X-100 were linear within the range from 50 to 2000 pg/ml. The total assay time was 3 h. Ultracentrifugation and immunoblot analysis indicated that human plasma and urine contained 50,000 g sedimentable and non-sedimentable forms of TF, both of which were detected by our ELISA method. Plasma and urine concentrations of TF in healthy subjects and patients with various diseases were measured by the ELISA method. In healthy subjects, plasma and urinary TF levels were found to be 149 +/- 72 pg/ml (n = 30) and 175 +/- 60 pg TF/urine creatinine mg (n = 95), respectively. TF was increased in plasma of patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, vasculitis associated with collagen diseases, diabetic microangiopathy and chronic renal failure receiving haemodialysis, but not in the plasma of endotoxaemic patients without DIC. The plasma TF/serum creatinine ratio did not show a positive correlation. Measurement of TF antigen in plasma may be useful for evaluating the endothelial damage and cell destruction in TF-containing tissues.

Adult↗
PubMed1994

Creatol, a creatinine metabolite, as a useful determinant of renal function.

Analysis of creatol (CTL, 5-hydroxycreatinine), an oxidative creatinine (Cr) metabolite, in serum and urine of human subjects has indicated that CTL is a useful determinant of renal function. The existence itself of serum CTL (s-CTL) could be a diagnostic sign for chronic renal failure (CRF): in all normal subjects, s-CTL was undetectable, but s-CTL was detectable in sera of all patients with CRF (s-Cr: > 2.0 mg/dl). And the s-CTL values increased in proportion to the severity of CRF in such patients. Furthermore, the molar ratio (CTL/Cr) in both urine and serum increased significantly in proportion to the severity of CRF. Our results indicated not only hyperproduction of CTL but also higher oxygen stress in patients according to the progression of CRF. The diagnostic importance of the CTL value and the CTL/Cr ratio are discussed.

Calibration↗
PubMed1994

Antitumor effect of recombinant human tumor necrosis factor on human testicular tumors heterotransplanted in nude mice.

The antitumor effect of recombinant human tumor necrosis factor (rHu-TNF) on 2 human testicular tumors (embryonal carcinoma, TTSC-1, and teratocarcinoma, TTSC-4) heterotransplanted in nude mice was studied. Therapeutic antitumor effect was observed more evidently in TTSC-1 than TTSC-4 when more than 5 x 10(3) U/mouse of rHu-TNF was administered intratumorally. Complete tumor regression was observed in both TTSC-1 and TTSC-4 after intratumoral administration of rHu-TNF. Histological findings correlated well with the decrease in tumor volume of treated tumors. In contrast to these data, little antitumor effect was observed when the same dose of rHu-TNF was administered intravenously to nude mice bearing these tumors. In conclusion, intratumoral administration of rHu-TNF was a more effective and safe method than intravenous administration against human testicular tumors heterotransplanted in nude mice.

Animals↗
PubMed1994

Monoclonal antibodies to a zinc-binding protein of rat Paneth cells.

Paneth cells are morphologically well characterized but their function has been not elucidated. Previously, we identified and purified a 90 KD zinc-binding protein (ZBPP-1) in rat intestine that was localized to Paneth cell granules, consistent with their high zinc content. To further elucidate the structure and function of ZBPP-1, we immunized Balb/c mice with purified ZBPP-1 and identified four independent monoclonal antibodies (MAb) producing MAb ZIP-1 (IgM), ZIP-2 (IgG1), ZIP-3 (IgM), and ZIP-4 (IgM). Immunohistochemistry (IHC) and immunoelectron microscopy (IEM) with these MAb showed positive staining of Paneth cell cytoplasmic granules. MAb ZBPP-1 also stained a population of mononuclear cells in the lamina propria of digestive tract mucosa and a few cells in spleen, presumably a subset of macrophages. These MAb will provide a useful tool to study the function of Paneth cells in human health and disease, since they cross-reacted with human intestinal Paneth cells and mucosal mononuclear cells.

Animals↗
PubMed1994

Studies on the mechanism controlling generation of superoxide radical in luteinized rat ovaries during regression.

The mechanism regulating the luteolytic release of superoxide radical (SOR) was examined in prostaglandin F2 alpha (PGF2 alpha)-treated rats. Tail vein injection of PGF2 alpha caused a rapid increase in SOR in mitochondria and plasma membrane samples prepared from luteinized rat ovaries. The peak in the mitochondria preceded that in the plasma membrane, and both occurred before progesterone concentrations decreased in the blood. The amount of SOR produced was greater when samples from the plasma membrane, mitochondria, and cytosol were combined. In plasma membrane samples, SOR generation was lowered by inhibitors of intracellular signaling pathways, but not by cyanide, which blocks electron transport in respiratory enzymes. In mitochondria samples, however, SOR was blocked by cyanide, but not by inhibitors of intracellular signaling enzymes. The addition of phospholipase-A2, phorbol myristate acetate (protein kinase-C activator), or arachidonic acid stimulated SOR production in plasma membrane samples from ovaries of control rats, and phorbol myristate acetate and arachidonic acid inhibited LH-stimulated progesterone secretion in dispersed rat luteal cells. Also, when mitochondria prepared from ovaries of PGF2 alpha-treated rats were added to dispersed corpus luteum cells, there was an increase in SOR generation and an inhibition of LH-stimulated cAMP formation and progesterone secretion. These results indicate that SOR production in the corpus luteum after PGF2 alpha treatment is generated by several subcellular components. Formation in the plasma membrane may be initiated by SOR generation from the mitochondria and regulated by intracellular signaling pathways. Our results indicate that formation of SOR may lead to the disruption of LH stimulation of progesterone secretion.

Animals↗
PubMed1994

Chemical modification of vesamicol binding sites and frequency augmentation-potentiation in neuromuscular transmission.

Vesamicol (AH5183), a potent and specific inhibitor of the vesicular acetylcholine uptake system, reduces at relatively high doses the slope of the log-linear frequency augmentation-potentiation (FAP) relation observed between the mean quantal content of endplate potential and the stimulation frequency. Recent biochemical studies identified two binding sites for vesamicol; one is the vesicular acetylcholine uptake system and the other is the vesamicol binding protein localized in the nerve terminal membrane. The present experiments were designed to determine the exact site of action of vesamicol on the FAP relation. Studies using epsilon-maleimidocaproic acid and picrylsulfonic acid, both membrane-impermeant protein-modifying reagents, revealed that these reagents diminished the vesamicol effect concomitant with an impairment of the functioning site for FAP. Because of the membrane-impermeant nature of the reagents, the present results were interpreted to mean that a vesamicol binding protein or a related component, localized in the nerve terminal membrane, is the target of action by vesamicol. This vesamicol binding component seems to play an important role in the stimulation frequency-dependent modulation of the evoked release of transmitter quanta observable as FAP.

Animals↗