Biomedical subjects

K Ohuchi

Publications and source records attributed to K Ohuchi.

At least 55 records · Page 3Linked to original sources

PubMed1998

Effects of glucocorticoids on apoptosis of infiltrated eosinophils and neutrophils in rats.

The effects of glucocorticoids on the survival of rat eosinophils and neutrophils infiltrated into the peritoneal cavity were examined. Glucocorticoids including dexamethasone, prednisolone and hydrocortisone inhibited the survival of rat peritoneal eosinophils at 10(-6) M, whereas they prolonged survival of rat peritoneal neutrophils at 10(-8) M. Sex steroids including estradiol and progesterone did not affect cell survival. Dexamethasone decreased the viability of eosinophils after 3 days of incubation and maintained the viability of neutrophils until 4 days after incubation concentration dependently. The EC50 of dexamethasone for inhibition of the survival of eosinophils was 1.5 x 10(-8) M, and that for the spontaneous death of neutrophils was 6.4 x 10(-10) M, suggesting that glucocorticoids at concentrations that inhibit eosinophil survival prolong neutrophil survival. Analysis of DNA fragmentation of cultured eosinophils and neutrophils revealed that glucocorticoids enhance eosinophil apoptosis but inhibit neutrophil apoptosis. The effects of dexamethasone on viability and DNA fragmentation were counteracted by the glucocorticoid receptor antagonist, mifepristone, concentration dependently. These findings indicate that glucocorticoids induce contradictory effects via the glucocorticoid receptor on rat eosinophils and neutrophils extravasated to an inflammatory locus such as the peritoneal cavity by modulating apoptosis.

Animals↗

PubMed1998

Identification of histamine-production-increasing factor produced by stimulated RBL-2H3 rat basophilic leukemia cells as granulocyte-macrophage colony-stimulating factor.

When RBL-2H3 rat basophilic leukemia cells were stimulated by antigen or the Ca2+ ionophore A23187, the activity to increase histamine production by rat bone marrow cells in the conditioned medium increased time-dependently. To characterize the histamine-production-increasing factor (HPIF) produced by RBL-2H3 cells, the conditioned medium was collected 8 h after stimulation by A23187, and the factor was purified by three-step chromatography, the specific activity being increased by 9000-fold. The partial amino acid sequence of the peptide obtained by S. aureus V8 protease digestion was identical to the internal amino acid sequence of rat granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, GM-CSF mRNA levels in RBL-2H3 cells were increased by A23187 with a peak at 4 h. Furthermore, recombinant rat GM-CSF increased histamine production by rat bone marrow cells. These findings suggested that HPIF produced by the stimulated RBL-2H3 cells is GM-CSF. Possible significant roles of HPIF at the late phase of allergic inflammation are discussed.

Amino Acid Sequence↗

PubMed1998

Analysis of the expression of intercellular adhesion molecule-1 in cells of the human bronchial epithelial cell line NCI-H292.

The mechanism of the expression of intercellular adhesion molecule-1 (ICAM-1) on epithelial cells was analyzed using NCI-H292 cells, a human bronchial epithelial cell line. Treatment with interferon-gamma (100 U/ml) or the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) (16.2 nM) induced ICAM-1 expression. The interferon-gamma-induced ICAM-1 expression was reduced by the tyrosine kinase inhibitor genistein (4',5,7-trihydroxyisoflavone) (37 to 185 microM), but not by the protein kinase C inhibitor Ro 31-8425 ((3-[8-(aminomethyl)-6,7,8,9-tetrahydropyrido [1.2-a]indol-10-yl]-4-(1-methyl-1 H-pyrrole-2,3-dione) (10 microM). The TPA-induced ICAM-1 expression was reduced by the protein kinase C inhibitor Ro 31-8425 (1 to 10 microM), but not by the tyrosine kinase inhibitor genistein (185 microM). The protein kinase A inhibitor H-89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide) did not affect the ICAM-1 expression induced by interferon-gamma or TPA. Pyrrolidine dithiocarbamate (1-pyrrolidinecarbodithioic acid) (100 microM), an inhibitor of nuclear factor kappaB (NF-kappaB) activation. enhanced the ICAM-1 expression induced by interferon-y, but reduced that induced by TPA. The changes in ICAM-1 expression on the cell surface were correlated with the changes in ICAM-1 mRNA levels. Combined treatment with interferon-gamma and TPA induced more than additive ICAM-1 expression. These findings suggest that interferon-gamma induces ICAM-1 expression by a tyrosine kinase-dependent mechanism, but that TPA induces it by a protein kinase C- and NF-kappaB-dependent mechanism.

Bronchi↗

PubMed1998

Effects of JTE-522, a specific inhibitor of cyclooxygenase-2, on the recurrence of allergic inflammation in rats.

JTE-522, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide , is a selective inhibitor of cyclooxygenase-2 at the enzyme level (IC50 is 6.4 x 10(-7) M for sheep cyclooxygenase-2, but it does not inhibit sheep cyclooxygenase-1 at concentrations up to 10(-4) M). In rat peritoneal macrophages in culture, it markedly inhibited cyclooxygenase-2-dependent prostaglandin E2 production and weakly inhibited cyclooxygenase-1-dependent prostaglandin E2 production, as did the selective cyclooxygenase-2 inhibitor NS-398 ([N-2(cyclohexyloxy-4-nitrophenyl)]-methanesulfonamide). In addition, the anti-inflammatory activity of JTE-522 was evaluated, using a model of recurrent air pouch-type allergic inflammation in rats. JTE-522, injected into the pouch just after a second antigen challenge, suppressed the accumulation of pouch fluid, the infiltration of leukocytes and the prostaglandin E2 content in the pouch fluid, as did NS-398 and indomethacin. These findings indicated that JTE-522 is a selective cyclooxygenase-2 inhibitor in cell culture systems and that the suppression by JTE-522 of the recurrence of allergic inflammation is due to the inhibition of cyclooxygenase-2.

Animals↗

PubMed1998

Inhibition of tumor necrosis factor-alpha production by SK&F 98625, a CoA-independent transacylase inhibitor, in cultured rat peritoneal macrophages.

When rat peritoneal macrophages were incubated in medium containing thapsigargin, tumor necrosis factor-alpha (TNF-alpha) production was increased time-dependently. In the presence of SK&F 98625, a CoA-independent transacylase inhibitor, the thapsigargin-induced TNF-alpha production was inhibited dose-dependently. Platelet-activating factor (PAF) and prostaglandin E2 (PGE2) production were also inhibited by SK&F 98625. The SK&F 98625-induced inhibition of TNF-alpha production was not prevented by addition of PGE2. PAF antagonists such as E6123, L-652,731 and CV-6209 partially inhibited the thapsigargin-induced TNF-alpha production, suggesting that concurrently produced PAF in thapsigargin-stimulated macrophages up-regulates TNF-alpha production. The inhibition by SK&F 98625 of thapsigargin-induced TNF-alpha production might be partly due to the inhibition of PAF production.

Acyltransferases↗

PubMed1998

Increase in histamine production by inflammatory exudate in the chronic phase of allergic inflammation in rats.

In the air pouch-type allergic inflammation in rats, we reported that a sustained histamine production in the late phase is induced by a cytokine-like factor, named histamine-production-increasing factor (HPIF) (1). Recently, we found another type of histamine-production-increasing factor in the pouch fluid at the chronic phase of air pouch-type allergic inflammation. Although it did not increase histamine production by itself, it enhanced the HPIF-induced histamine production by rat bone marrow cells. It also increased GM-CSF-induced histamine production. The activity of this factor increased time-dependently from 3 to 7 days after the antigen challenge. Injection of the 5 day pouch fluid sample containing this factor into the pouch 4 h after the antigen challenge increased histamine contents in the pouch fluid at 24 h, indicating that this factor enhances HPIF-induced histamine production in vivo. Biochemical analysis of the 5 day pouch fluid sample indicated that this factor is a heat-labile and trypsin-sensitive protein of which pI value and molecular weight are 7-8 and about 100 kDa, respectively.

Animals↗

PubMed1998

A model analysis of aftereffects of high-intensity DC stimulation on action potential of ventricular muscle.

The mechanism for aftereffects of high-intensity dc stimulation on ventricular muscle was studied by using Beeler-Reuter's action potential model. A leak conductance (Gpore, maximal value from 40 to 80 microS for 1 cm2 of membrane), which mimics reversible dielectric breakdown of the cell membrane by the shock, was incorporated into the model. To simulate resealing process, Gpore was assumed to decrease after the shock exponentially at a time constant (tau pore) of 5-50 s. The simulation results are qualitatively consistent with our experimental observations in guinea pig papillary muscle [1]; they include prolonged depolarization, diastolic depolarization or oscillation of membrane potential leading to a single or multiple spontaneous excitation. The phase-independence and shock intensity-dependence can also be reproduced. Analysis of current components has revealed that: 1) a large inward leak current (Ileak) is responsible for the prolonged depolarization; 2) time-dependent decay of outward current (IX1) in combination with Ileak and slow inward current (I(s)) results in diastolic depolarization or oscillation of membrane potential; 3) spontaneous excitation depends on an activation of I(s). These findings support our hypothesis that strong shocks (> 15 V/cm) will produce abnormal arrhythmogenic responses in ventricular muscle through a transient rupture of sarcolemmal membrane.

Action Potentials↗

PubMed1998

Expression of adhesion molecules in cultured human nasal mucosal microvascular endothelial cells activated by interleukin-1 beta or tumor necrosis factor-alpha: effects of dexamethasone.

Adhesion molecules of microvascular endothelial cells play a key role in the inflammatory processes involved in nonallergic sinusitis. We investigated the cytokine-regulated expression of intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1 (VCAM-1), and the effect of dexamethasone on these expressions in cultured human nasal microvascular endothelial cells (HNMEC). ICAM-1 was enhanced, and E-selectin and VCAM-1 were induced in a dose-dependent fashion following stimulation with IL-1beta or TNF-alpha. HNMEC differed from human umbilical vein endothelila cells in that (1) maximal upregulation of ICAM-1 expression induced by IL-1beta or TNF-alpha required more time (2) TNF-alpha was more potent than IL-1beta in VCAM-1 expression, and (3) dexamethasone inhibited the upregulation of E-selectin expression alone. These findings contribute to a better understanding of the characteristic features of leukocyte infiltration into inflamed tissue and the effect of glucocorticoid in nonallergic chronic sinusitis.

Cell Adhesion Molecules↗

PubMed1998

Identification of cDNA for rat homologues of human major basic protein and eosinophil cationic protein.

We have determined the complete nucleotide sequence for cDNA of rat homologues of human eosinophil major basic protein (MBP) and eosinophil cationic protein (ECP) using the rapid amplification of cDNA ends (RACE) procedure. Nucleotide sequence of cDNA of rat MBP revealed that mRNA of rat MBP encodes a protein containing 227 amino acids which has three functional domains; namely, the signal peptide, the acidic peptide that contains numerous acidic amino acids and the mature MBP, as in human, guinea pig and mouse MBP. In addition, cDNA of a rat homologue of human ECP was also cloned. The deduced amino acid sequence revealed that this gene encodes a putative protein with a molecular weight of 15.5 kD which has ribonuclease activity. The homology of amino acid sequence between the rat homologue and the murine eosinophil-associated ribonucleases (EARs) was high (65%). Therefore, we named this rat homologue 'rat EAR-1'.

Amino Acid Sequence↗

PubMed1997

Induction of nitric oxide synthase by lipopolysaccharide and its inhibition by auranofin in RAW 264.7 cells.

In RAW 264.7 cells, a murine macrophage cell line, treatment with lipopolysaccharide (1 to 10 ng/ml) stimulated production of nitric oxide (NO), which was inhibited by L-N(G)-monomethyl-L-arginine acetate, an inhibitor of NO synthase. Auranofin, an orally active chrysotherapeutic agent, also inhibited the lipopolysaccharide-induced NO production in a concentration-dependent manner (0.3 to 3 microM). Other gold salts such as aurothioglucose and aurothiomalate had no effect. Western blot analysis demonstrated that the lipopolysaccharide (10 ng/ml)-induced expression of inducible NO synthase protein was inhibited by auranofin as well as by the protein synthesis inhibitor cycloheximide. In addition, the lipopolysaccharide-induced increase in the level of mRNA for inducible NO synthase was also lowered by auranofin. Furthermore, auranofin showed no direct effect on the conversion of [3H]arginine to [3H]citrulline by the cell lysate. These findings indicate that auranofin inhibits lipopolysaccharide-induced NO production by suppressing the expression of inducible NO synthase.

Animals↗

PubMed1997

Possible participation of macrophage inflammatory protein 2 in neutrophil infiltration in allergic inflammation in rats.

Recombinant rat macrophage inflammatory protein 2 (MIP-2) was prepared from E. coli transfected with a glutathione-S-transferase (GST)-MIP-2 fusion protein expression vector. A polyclonal antibody to rat MIP-2 was then obtained from rabbits by immunization with recombinant rat MIP-2. Using the polyclonal antibody which selectively suppressed neutrophil chemotactic activity of MIP-2, the role of MIP-2 in neutrophil infiltration in allergic inflammation in rats was studied. In an air pouch-type allergic inflammation model in rats, neutrophil infiltration into the pouch fluid increased with time after antigen challenge. Neutrophil chemotactic activity in the pouch fluid collected 8 h after antigen challenge was diminished by anti-MIP-2 antibody. In addition, when leukocytes that had infiltrated into the pouch fluid collected 4 h after antigen challenge were incubated, neutrophil chemotactic activity in the conditioned medium increased time-dependently, and the activity was neutralized by anti-MIP-2 antibody. Furthermore, when anti-MIP-2 antibody was injected into the pouch 6 h after antigen challenge, neutrophil infiltration into the pouch fluid during the next 2 h was suppressed. These findings indicate that MIP-2 plays an important role in neutrophil infiltration in rat allergic inflammation.

Amino Acid Sequence↗

PubMed1997

Dual effects of auranofin on prostaglandin E2 production by rat peritoneal macrophages.

Incubation of rat peritoneal macrophages in medium containing various concentrations of auranofin (1, 3 and 10 microM) increased prostaglandin E2 production at 4 h in a concentration-dependent manner, in accordance with the increase in the release of [3H]arachidonic acid from membrane phospholipids. However, at 20 h, no stimulation of prostaglandin E2 production by auranofin was observed. When the peritoneal macrophages were incubated in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), thapsigargin or A23187, prostaglandin E2 production at 4 and 20 h was enhanced. The stimulator-induced prostaglandin E2 production at 20 h was suppressed by 10 microM of auranofin. Western blot analysis demonstrated that auranofin inhibited the induction of cyclooxygenase 2 by TPA, thapsigargin or A23187 at 4 and 20 h. The level of cyclooxygenase 1 did not change by treatment with these stimulators in the presence or absence of auranofin. These findings suggest that auranofin has dual effects on prostaglandin E2 production: without stimulation, auranofin increases prostaglandin E2 production at 4 h due to the increased release of arachidonic acid which is converted to prostaglandin E2 mainly by cyclooxygenase 1, but inhibits the stimulator-induced late-phase prostaglandin E2 production by inhibiting the induction of cyclooxygenase 2.

Animals↗

PubMed1997

Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease.

Using the rapid amplification of cDNA ends (RACE) procedure, we have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil cationic protein (ECP)/eosinophil-associated ribonuclease (EAR). The deduced amino acid sequence revealed that the molecular weight of rat preECP/EAR is 18.0 kDa and the isoelectric point is 9.85, indicating that rat ECP/EAR is highly cationic. The homology of amino acid sequence between rat ECP/EAR and human ECP is 54%, and that between rat ECP/EAR and human eosinophil-derived neurotoxin (EDN) is 51%. Rat ECP/EAR is also homologous to human ribonuclease k6 (homology 47%).

Amino Acid Sequence↗

PubMed1997

Possible participation of cyclooxygenase-2 in the recurrence of allergic inflammation in rats.

In the recurrence of allergic inflammation in a rat air pouch model, pouch fluid volume, prostaglandin E2 concentration in the pouch fluid, leukocyte infiltration into the pouch fluid, and granulation tissue weight were markedly increased by the antigen challenge. To clarify the role of cyclooxygenase-2 in the recurrence of allergic inflammation, the time-course of changes in protein levels of cyclooxygenase-1 and cyclooxygenase-2 in the granulation tissue and in the infiltrated leukocytes was examined by Western blot analysis. It was shown that cyclooxygenase-1 levels in the granulation tissue and in the infiltrated leukocytes were not changed by the antigen challenge, but cyclooxygenase-2 levels were increased. Furthermore, treatment with the selective cyclooxygenase-2 inhibitor, NS-398 ([N-2(cyclohexyloxy-4-nitrophenyl]-methanesulfonamide), suppressed the recurrence of allergic inflammation as did the non-selective cyclooxygenase-1/cyclooxygenase-2 inhibitor, indomethacin. The steroidal anti-inflammatory drug, dexamethasone, inhibited the induction of cyclooxygenase-2, and suppressed the allergic inflammation. These findings strongly suggested that cyclooxygenase-2 induced by the antigen challenge plays a role in the recurrence of inflammation induced by the allergic mechanism.

Animals↗

PubMed1997

Mercury dynamics in hair of rats exposed to methylmercury by synchrotron radiation X-ray fluorescence imaging.

Two-dimensional distribution of mercury (Hg) in hair samples of rats exposed to methylmercury (MeHg) was analyzed by synchrotron radiation X-ray fluorescence (SR-XRF) imaging. Experiments with endogenous- and exogenous-model for MeHg exposure revealed that the metal level was obviously higher in the hair cortex after the former exposure whereas a dominant site that Hg distributed after the latter exposure was the cuticle. The method also provided us the Hg profile along the hair length with a single hair obtained by the endogenous model. Thus application of SR-XRF analysis to hair sample would facilitate biological monitoring to not only distinct Hg exposure but also determine its dynamics with only the specimen.

Animals↗

PubMed1997

Pharmacological analysis of the inflammatory exudate-induced histamine production in bone marrow cells.

The inflammatory exudate at the post-anaphylaxis phase of allergic inflammation in rats has an ability to enhance histamine production by bone marrow cells. To analyze the mechanism of the inflammatory exudate-induced histamine production pharmacologically, the effects of several drugs were examined in cultures of bone marrow cells. Incubation of the bone marrow cells in the presence of the inflammatory exudate that had been centrifuged and dialyzed against Hanks' balanced salt solution increased histidine decarboxylase activity in the cells and histamine concentration in the conditioned medium. The induction of histamine production by the inflammatory exudate was inhibited by actinomycin D (0.01-1 microM), an inhibitor of RNA synthesis, and cycloheximide (0.1-10 microM), a protein synthesis inhibitor. The protein kinase C inhibitors staurosporine (2-20 nM), K-252a (6-200 nM), and H-7 (10.3-103 microM) also inhibited the inflammatory exudate-induced histamine production in a concentration-dependent manner. The tyrosine kinase inhibitor genistein (3.7-37 microM) also inhibited the inflammatory exudate-induced histamine production, but the protein kinase A inhibitor H-89 (0.2 microM), and the adenylate cyclase activator forskolin (0.1 microM) showed no effect. These findings suggest that histamine production induced by the inflammatory exudate is mediated by the de novo synthesis of histidine decarboxylase and by the activation of protein kinase C and tyrosine kinase.

Anaphylaxis↗

PubMed1997

Induction of neutrophil chemotactic factor production by staurosporine in rat peritoneal neutrophils.

1. Incubation of rat peritoneal neutrophils in medium containing various concentrations of staurosporine (6.4-64 nM) increased the neutrophil chemotactic activity in the conditioned medium in a time- and concentration-dependent manner. 2. Separation of the neutrophil chemotactic activity in the conditioned medium by isoelectric focusing revealed that staurosporine (64 nM) stimulated the production of basic (pH > 8) neutrophil chemotactic factors, while TPA (12-O-tetradecanoylphorbol 13-acetate, 49 nM) stimulated the production of both basic (pH > 8) and acidic (pH 5) neutrophil chemotactic factors. 3. Determination by immunoassay of cytokine-induced neutrophil chemoattractant (CINC)-1, -2 alpha, -2 beta and -3 in the conditioned medium at 4 h revealed that staurosporine (64 nM) and TPA (49 nM) strongly stimulated the production of CINC-3 (staurosporine, 133.0 +/- 3.8; TPA, 26.7 +/- 1.0; control, 0.32 +/- 0.01 ng ml-1, means +/- s.e.mean from four samples) compared to CINC-1 (staurosporine, 55.0 +/- 1.2; TPA, 12.2 +/- 0.3; control, 0.56 +/- 0.01 ng ml-1), and CINC-2 alpha (staurosporine, 1.09 +/- 0.03; TPA, 0.90 +/- 0.02; control, < 0.10 ng ml-1). CINC-2 beta was below the detectable amount (< 0.078 ng ml-1). 4. The level of CINC-3 mRNA in the peritoneal neutrophils was determined by reverse transcription-polymerase chain reaction. Staurosporine (64 nM) and TPA (49 nM) enhanced the level of CINC-3 mRNA time-dependently, but had no effect on GAPDH mRNA levels. 5. Production of staurosporine-induced neutrophil chemotactic factor was inhibited by the protein kinase C inhibitors, H-7 (IC50, 12.3 microM), calphostin C (IC50, 0.77 microM) and Ro 31-8425 (24.3% inhibition at 10 microM), and by the tyrosine kinase inhibitor, genistein (IC50, 68.5 microM). Production of TPA-induced neutrophil chemotactic factor was also inhibited by both inhibitors. 6. Both the staurosporine- and the TPA-induced increase in CINC-3 mRNA levels were suppressed by H-7 and genistein.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Increase of eosinophilic cell population in bone marrow of rats by immunization with Ascaris suum antigen.

Immunization of rats with the antigen, Ascaris suum extract, increased the number of peripheral eosinophils. Analysis by Western blot and reverse transcription-polymerase chain reaction revealed that the levels of major basic protein and its mRNA in the bone marrow were also increased, suggesting that eosinophilic cell population in the bone marrow is increased by the immunization. These findings indicate that immunization with this antigen stimulates differentiation of progenitor cells to eosinophils in the bone marrow, and induces blood eosinophilia.

Animals↗