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Biomedical subjects

M Murakami

Publications and source records attributed to M Murakami.

At least 613 records · Page 34Linked to original sources
PubMed1996

Inducible expression of glial fibrillary acidic protein in HT-1080 human fibrosarcoma cells.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed almost exclusively by glial cells of the central nervous system. We have previously transfected GFAP-negative human astrocytoma cells with the gene for GFAP and have demonstrated that GFAP transfection decreases astrocytoma proliferation and alters astrocytoma morphology. To determine if the same cellular responses could be elicited upon GFAP transfection of nonglial tumor cells, in the present study we have transfected a GFAP-negative human malignant fibrosarcoma cell line (HT-1080) with a cDNA containing the entire coding sequence of the human GFAP gene under the control of an inducible metallothionein promoter. Stably transfected HT-1080 clones were identified that are GFAP-positive by PCR and immunocytochemistry. GFAP-positive HT-1080 fibrosarcoma cells also demonstrate a decrease in tumor cell proliferation, altered morphological features characterized by cell elongation and cytoplasmic process formation, and reduction of invasive potential when compared to controls. These findings suggest that the inducible expression of the cytoskeletal protein GFAP can also be associated with dramatic cellular effects in nonglial non-central nervous system tumor cells.

Agar↗
PubMed1996

Effect of lansoprazole in mono-, dual-, or triple therapy on Helicobacter pylori eradication.

The effect of lansoprazole, in mono, dual, or triple therapy, on the eradication of Helicobacter pylori was reviewed. Lansoprazole has a cytotoxic action against this organism, the MIC being 2.56-5.25 micrograms/ml. In in vitro experiments, lansoprazole exerts direct action, i.e., antibacterial activity of lansoprazole against H. pylori and also inhibits urease activity. With antibiotics, this drug has a synergistic effect against H. pylori. In clinical studies, the eradication rate of H. pylori with lansoprazole is 0%-25% with monotherapy, 22%-33% with dual therapy (of AMPC), and 75%-82.4% with triple therapy with metronidazole and antibiotics. We inves-tigated the effect of lansoprazole on the eradication of H. pylori with dual therapy, the other agent being amoxicillin (AMPC). The eradication rate was 0% when 30 mg lansoprazole was employed as monotherapy, 33% for dual therapy with 30 mg lansoprazole and 1 g AMPC, and 71% for dual therapy with 60 mg lansoprazole and 1 g AMPC, the eradication rate with 60 mg lansoprazole and 1 g AMPC being significantly higher than that with the lower dose of lansoprazole. This result suggested that the greater acid suppression brought about by lansoprazole 60 mg enhances the action of AMPC, indicating that lansoprazole, when used with AMPC, is effective for eradication of H. pylori.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1995

Regulation of rat liver type 1 iodothyronine deiodinase mRNA levels by testosterone.

To investigate the underlying mechanisms of sex-related differences in liver type 1 iodothyronine deiodinase (ID1), we studied the sex-related differences and roles of sex steroids in liver ID1 mRNA levels in the rat. In both euthyroid and thyroidectomized rats, liver ID1 activity and ID1 mRNA levels in female rats were less than those in male rats. A positive correlation was observed between liver ID1 activity and ID1 mRNA levels. Liver ID1 activity and ID1 mRNA levels in male rats decreased after orchiectomy, and were increased to control levels by testosterone administration. Ovariectomy of beta-estradiol administration did not alter liver ID1 activity or ID1 mRNA levels in female rats. ID1 mRNA levels in cultured rat hepatocytes were significantly increased by testosterone, but not by beta-estradiol. These results suggest that the sex-related differences in liver ID1 activity are attributable to differences in ID1 mRNA levels, and that testosterone plays an important role in the sex-related differences in liver ID1 mRNA levels.

Animals↗
PubMed1995

IgE-dependent activation of cytokine-primed mouse cultured mast cells induces a delayed phase of prostaglandin D2 generation via prostaglandin endoperoxide synthase-2.

Mouse bone marrow-derived mast cells (BMMC) developed with IL-3 generate prostaglandin D2 (PGD2) through the utilization of prostaglandin endoperoxide synthase (PGHS)-1 within several minutes of cross-linking the high affinity Fc receptor for IgE (Fc epsilon RI) by hapten-specific IgE and Ag. We now report that this immediate generation of PGD2 is followed by a 15-fold induction of steady-state transcripts for PGHS-2, with a maximum at 30 min, accompanied by transient expression of PGHS-2 protein. When BMMC were pretreated with c-kit ligand (KL) in combination with IL-10 for 2 h, sensitized with IgE, and activated with Ag, their expression of steady-state transcripts for PGHS-2 increased 111-fold and their expression of PGHS-2 protein was markedly enhanced, with maximal expression at 1 h and 5 h, respectively, after activation. These events were accompanied by PGD2 generation from 1 to 10 h after activation that accounted for approximately 50% of total PGD2 generation. The expression of PGHS-1 protein did not change during this period. The optimal priming interval for the effect of KL plus IL-10 on the IgE-dependent induction of PGHS-2 was 2 h, at which time only this particular cytokine combination acted synergistically with activation by IgE and Ag. In contrast, at 2 days the accessory cytokines that could provide priming with KL included IL-3 and IL-9 in addition to IL-10. Dexamethasone, which inhibited the expression of PGHS-2 but not PGHS-1, and NS-398, a selective inhibitor of PGHS-2, each suppressed the delayed phase but not the immediate phase of PGD2 generation. Conversely, valeryl salicylate, a selective inhibitor of PGHS-1, suppressed the immediate but not the delayed phase of PGD2 generation after cell priming and IgE-dependent activation.

Animals↗
PubMed1995

Interleukin-3 regulates development of the 5-lipoxygenase/leukotriene C4 synthase pathway in mouse mast cells.

To study cytokine regulation of the 5-lipoxygenase (5-LO)/leukotriene (LT) synthase pathway we have developed mouse bone marrow-derived mast cells (BMMC) that minimally express each protein of the pathway by using a novel culture system, lacking interleukin (IL)-3. When mouse bone marrow cells were cultured for 5 weeks with 100 ng/ml c-kit ligand (KL) and 10 units/ml IL-10, a population of > 95% mast cells was obtained. These cells generated 8.3 +/- 4.5 ng of LTC4/10(6) cells and 8.1 +/- 2.4 ng of prostaglandin (PG) D2/10(6) cells after IgE-dependent activation. When these BMMC were cultured for 2-5 weeks more with 100 units/ml IL-3 in the continued presence of KL and IL-10, the IgE-dependent generation of LTC4 and PGD2 increased to 212 +/- 36 and 25.5 +/- 8.6 ng/10(6) cells, respectively. The dramatic increase in the IgE-dependent generation of LTC4 in response to IL-3 was accompanied by a concomitant increase in expression of 5-LO and 5-LO-activating protein and preceded the increased expression of cytosolic phospholipase A2 and LTC4 synthase. The recognition that IL-3 up-regulates the expression of each protein of the 5-LO pathway for the generation of LTC4 contrasts with our recent finding that KL up-regulates the expression of cytosolic phospholipase A2, prostaglandin endoperoxide synthase-1, and hematopoietic PGD2 synthase and increases the IgE-dependent generation of PGD2 in BMMC developed from bone marrow with IL-3. Thus, developmentally segregated regulation of the prostanoid and cysteinyl leukotriene pathways in lineage-related committed mast cell progenitors reveals the pleiotropism of this effector cell of allergic inflammation, a cytokine/growth factor basis for preferential expression of pathways of eicosanoid biosynthesis, and the particular role of IL-3 in regulating the expression of the proteins of the 5-LO/LTC4 synthase pathway.

Animals↗
PubMed1995

Activation of the thyrotropin-releasing hormone (TRH) receptor by a direct precursor of TRH, TRH-Gly.

We studied the mechanism by which thyrotropin-releasing hormone (TRH)-Gly stimulated prolactin and thyrotropin (TSH) secretion in pituitary, using a pituitary mammotropic cell line, GH3 cells, and a cell line stably expressing a human TRH receptor (TRH-R). In GH3 cells expressing endogenous TRH-R, an addition of TRH-Gly evoked an immediate rise of intracellular calcium concentration, indicating that TRH-Gly reacted directly without converting from TRH-Gly to TRH. In order to determine whether this reaction might occur through TRH-R, we established a cell line stably expressing a human TRH-R, by transfecting a human TRH-R cDNA into Chinese hamster ovary cells (CHO cells). In this cell line, 10 nM TRH elevated intracellular calcium significantly; the Kd for MeTRH was 1.7 nM. One micromolar and 100 nM TRH-Gly also elevated intracellular concentration of calcium significantly, but not in CHO cells which were not transfected with the TRH-R cDNA. Competition studies further revealed that TRH-Gly displaced MeTRH binding (IC50, 12 microM). These data indicate that at high concentration, TRH-Gly interacts directly with TRH-R to activate signal transduction pathway, and that release of prolactin and TSH induced by TRH-Gly in vitro may be due, at least in part, to the direct effect of TRH-Gly on the TRH-R.

Animals↗
PubMed1995

The immediate phase of c-kit ligand stimulation of mouse bone marrow-derived mast cells elicits rapid leukotriene C4 generation through posttranslational activation of cytosolic phospholipase A2 and 5-lipoxygenase.

c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.

Animals↗
PubMed1995

Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells.

Mouse bone marrow-derived mast cells (BMMCs) developed with interleukin 3 (IL-3) can be stimulated by c-kit ligand (KL) and accessory cytokines over a period of hours for direct delayed prostaglandin (PG) generation or over a period of days to prime for augmented IgE-dependent PG and leukotriene (LT) production, as previously reported. We now report that IL-4 is counterregulatory for each of these distinct KL-dependent responses. BMMCs cultured for 4 days with KL + IL-3 or with KL + IL-10 produced 5- to 7-fold more PGD2 and approximately 2-fold more LTC4 in response to IgE-dependent activation than BMMCs maintained in IL-3 alone. IL-4 inhibited the priming for increased IgE-dependent PGD2 and LTC4 production to the level obtained by activation of BMMCs maintained in IL-3 alone with an IC50 of approximately 0.2 ng/ml. IL-4 inhibited the KL-induced increase in expression of cytosolic phospholipase A2 (cPLA2) but had no effect on the incremental expression of PG endoperoxide synthase 1 (PGHS-1) and hematopoietic PGD2 synthase or on the continued baseline expression of 5-lipoxygenase, 5-lipoxygenase activating protein, and LTC4 synthase. BMMCs stimulated by KL + IL-10 for 10 h exhibited a delayed phase of PGD2 generation, which was dependent on de novo induction of PGHS-2. IL-4 inhibited the induction of PGHS-2 expression and the accompanying cytokine-initiated delayed PGD2 generation with an IC50 of approximately 6 ng/ml. IL-4 had no effect on the expression of PGHS-2 and the production of PGD2 elicited by addition of IL-1 beta to the combination of KL + IL-10. IL-4 had no effect on the immediate phase of eicosanoid synthesis elicited by KL alone or by IgE and antigen in BMMCs maintained in IL-3. Thus, the counterregulatory action of IL-4 on eicosanoid generation is highly selective for the induced incremental expression of cPLA2 and the de novo expression of PGHS-2, thereby attenuating time-dependent cytokine-regulated responses to stimulation via Fc epsilon receptor I and stimulation via c-kit, respectively.

Animals↗
PubMed1995

Neurochemical changes related to ageing in the senescence-accelerated mouse brain and the effect of chronic administration of nimodipine.

The levels of neurotransmitters and related metabolic enzyme activities in the brain of young-adult (3 months old), aged (11 months old) and nimodipine-administered (11 months old) senescence-accelerated mouse (SAM) were compared. Nimodipine, a calcium antagonist, was administered orally for 5 months. Acetylcholine (ACh), serotonin (5-HT) and dopamine (DA) levels all decreased with age but this decrease was attenuated by nimodipine. Choline acetyltransferase and choline esterase activities increased with age, and nimodipine enhanced their activities. Tryptophan hydroxylase activity was not affected by age or nimodipine administration. Monoamine oxidase-A activity increased with age, and was decreased by nimodipine administration. These results suggest that SAM rapidly undergoes neurochemical changes which are considered to be part of the normal aging process, and these changes were attenuated by chronic administration of nimodipine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Effect of chronic L-dopa administration on serum luteinizing hormone levels in male rats.

We examined whether the repeated oral administration with a high dose of L-3-(3,4-dihydroxyphenyl)-alanine (L-DOPA) in 0.5% carboxymethyl cellulose increases serum luteinizing hormone (LH) levels in male rats. Serum LH levels were increased 4 h after a single administration of 1000 mg/kg L-DOPA to male rats, and returned to control levels within 8 h after administration. Four hours after a single administration, serum LH levels were significantly increased by L-DOPA at 1000 mg/kg, but not at 20, 100 or 200 mg/kg. Decreases in body weight and relative weight of the prostate were observed after 7 and 14 days of administration of 1000 mg/kg per day L-DOPA, but no changes were observed in weight of the testis, epididymis or seminal vesicle. The administration of L-DOPA at 500 or 1000 mg/kg per day for 7 or 14 days resulted in increased basal serum LH levels and decreased basal serum prolactin levels 24 h after the last administration. Serum testosterone levels tended to be higher in treated than in control rats. The levels of two metabolites of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in rats treated with 500 mg/kg per day tended to be slightly higher than those in control rats after 7 days of administration. Levels of DA, DOPAC and HVA were significantly increased after 7 and 14 days of administration of 1000 mg/kg per day and after 14 days of administration of 5000 mg/kg per day. The level of norepinephrine, but not its metabolite 3-methoxy-4-hydroxyphenylglycol, was significantly increased after only 7 days of administration of 1000 mg/kg per day. No significant changes were observed in levels of 5-hydroxytryptamine or its metabolite 5-hydroxyindole-3-acetic acid with administration of 500 or 1000 mg/kg per day. These findings suggest that a prolonged treatment with a high dose of L-DOPA in male rats induces release of LH from the pituitary, resulting in sustained elevation of LH levels in peripheral circulation.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Reducing interference from heterophilic antibodies in a two-site immunoassay for carcinoembryonic antigen (CEA) by using a human/mouse chimeric antibody to CEA as the tracer.

To reduce heterophilic antibody interference in a two-site immunoassay for carcinoembryonic antigen (CEA), we utilized a human/mouse chimeric antibody to CEA as the tracer. One mouse monoclonal antibody (MAb), F82-61, which reacts with an epitope present on the domain N of CEA, was immobilized on 96-well polystyrene microtiter plates. A human/mouse chimeric antibody (Ch F11-39), which recognizes an epitope present on the domain B3 of CEA, was biotinylated for the tracer (Ch F11-39 system). Another MAb F11-39, the parental MAb of Ch F11-39, was also biotinylated and used as the control tracer (F11-39 system). For a fair comparison, the same 503 serum samples from healthy individuals were simultaneously assayed in the present study. When a tentative common reference limit of 5 ng/ml was used, the false positive rate with the Ch F11-39 system was only 2.8% (14/503) and that with the F11-39 system was 29.0% (146/503). Adding normal mouse serum (NMS; 1%) or a mixture of purified mouse IgG subclasses (heterophilic blocking reagent (HBR, 15 micrograms/test)) to the F11-39 system reduced the false positive rate from 29.0% to 6.2% (31/503) or 4.8% (24/503), respectively, suggesting that heterophilic antibodies reactive with mouse IgG gave rise to the high positive rate in normal populations with the F11-39 system. On the other hand, the false positive rate with the Ch F11-39 system was only slightly reduced from 2.8% to 2.6% (13/503) or to 2.0% (10/503) by adding NMS or HBR to the Ch F11-39 system. The false positive rates with two commercially available assay systems, CEA Roche EIA.DM or Abbott IMx CEA, were 5.4% (27/503) and 5.8% (29/503), respectively, which both corresponded roughly to that with the F11-39 system including NMS or HBR. These results indicate that the application of human/mouse chimeric antibodies in two-site immunoassays is more effective for reducing interference from heterophilic antibodies than the adding of NMS or purified mouse IgG in the assay using conventional MAbs.

Animals↗