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Biomedical subjects

K Ohuchi

Publications and source records attributed to K Ohuchi.

At least 91 records · Page 5Linked to original sources
PubMed1994

Possible roles of protein kinases in neutrophil chemotactic factor production by leucocytes in allergic inflammation in rats.

1. In an air pouch-type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2. To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3. When the infiltrated leucocytes were incubated for 4 h in medium containing the non-selective protein kinase inhibitor K-252a (1-100 ng ml-1, 2.14-214 nM), the tyrosine kinase inhibitor genistein (1-50 micrograms ml-1, 3.7-185 microM), and the more selective protein kinase C inhibitor H-7 (5-100 micrograms ml-1, 13.7-274 microM); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration-dependent manner, but the adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor H-89 (1-1000 ng ml-1, 2.24-2240 nM) showed no effect. 4. Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte-derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF-2. Treatment of the leucocytes with K-252a, genistein, and H-7, but not H-89, inhibited production of both LDNCF-1 and LDNCF-2. 5. These results suggest that activation of tyrosine kinase and protein kinase C, but not cAMP-dependent protein kinase, is responsible for the production of LDNCF-1 and LDNCF-2. 6. The steroidal anti-inflammatory drug dexamethasone and the protein synthesis inhibitor cycloheximide inhibited neutrophil chemotactic factor production in a concentration-dependent manner. Time-course experiments showed that the inhibitory effect by dexamethasone was apparent even 30 min after the incubation.7. Mechanism for inhibiting the production of LDNCF-1 and LDNCF-2 by dexamethasone is also discussed.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

Inhibition of histamine release from RBL-2H3 cells by protein synthesis inhibitors.

Effects of cycloheximide, an inhibitor of protein synthesis, on histamine release from RBL-2H3 cells were examined. RBL-2H3 cells sensitized by rat antiserum to ascaris extract were challenged by the antigen, and histamine release during a period of 30 min was measured. Pretreatment with cycloheximide (1 microgram/ml) for 1 h significantly inhibited the antigen-induced histamine release (36% inhibition). The cycloheximide-induced inhibition of histamine release was abolished when the cells were further incubated in the absence of cycloheximide for 2 h. Pretreatment with puromycin (3 and 10 micrograms/ml), an inhibitor of protein synthesis, or actinomycin D (0.1-1 microgram/ml), an inhibitor of DNA-dependent RNA synthesis, also inhibited the antigen-induced histamine release in a concentration-dependent manner. Both ionomycin- and thapsigargin-induced histamine release were also inhibited by pretreatment with cycloheximide. Measurement of intracellular Ca2+ levels using quin 2 revealed that cycloheximide inhibits the increase in Ca2+ levels induced by the antigen, ionomycin or thapsigargin. These results suggest that histamine release induced by the antigen, ionomycin and thapsigargin in RBL-2H3 cells is mediated by protein(s) which is newly synthesized and inactivated rapidly, and the newly synthesized protein(s) is involved in the increase of intracellular Ca2+ levels induced by these stimulants.

Animals↗
PubMed1993

Stimulation of prostaglandin E2 production and induction of specific protein synthesis in rat peritoneal macrophages by a tumor promoter staurosporine.

Staurosporine is a microbial anti-fungal alkaloid having potent inhibitory activity on protein kinase C and is a non 12-O-tetradecanoylphorbol-13-acetate-type tumor promoter in two-stage carcinogenesis experiments in mouse skin. Effects of staurosporine and its structurally related compounds K-252a, KT5720 and KT5822 on prostaglandin E2 production, release of arachidonic acid from membrane phospholipids, and uptake of [35S]methionine into intracellular proteins were examined in rat peritoneal macrophages. Among the four compounds, only staurosporine stimulated the production of prostaglandin E2 and release of arachidonic acid at concentrations of 1 ng/ml and 10 ng/ml. The uptake of [35S]methionine into cellular proteins, estimated to be 120 kDa and 125 kDa molecular mass, was also stimulated by staurosporine treatment, and the uptake was increased in parallel with the increase in prostaglandin E2 production. At higher concentrations (100 ng/ml and 1000 ng/ml), staurosporine inhibited prostaglandin E2 production and did not induce the specific protein synthesis. Other compounds neither stimulated prostaglandin E2 production nor induced specific protein synthesis. K-252a inhibited prostaglandin E2 production at concentrations above 10 ng/ml. These results suggest that the staurosporine-induced proteins might participate in the tumor promotion or at least in the staurosporine-induced stimulation of prostaglandin E2 production.

Alkaloids↗
PubMed1993

Some features in prostaglandin synthesis of the cancer cells which metastasized into liver from intestinal cancer lesions.

In order to study the mechanism of cancer metastasis, AH100B cells, an ascitic hepatoma cell line, were transplanted into the small intestine of male Donryu rats. Each metastatic nodule in the liver was collected with the respective intestinal lesion. Each sample thus obtained was injected into the peritoneal cavity of male Donryu rats to make free cancer cells. Then, the cancer cells, having an intact cell surface, of the metastatic and primary intestinal lesion were collected respectively. After washing in Dolbecco's PBS (Ca2+ and Mg(2+)-free, pH 7.2), the definite numbers of cancer cells of the metastatic and primary intestinal lesion were incubated in the PBS containing [1-14C]-AA at 25 degrees C for 30 min, respectively. AA metabolites formed during the incubation period were extracted and subjected to TLC, followed by autoradiography. Each radioactive part was scraped off the plate and measured for its radioactivity. The pattern of the ability to synthesize PGs was different between the cancer cells which metastasized to the liver and those of the primary lesion, that is, percentage values of PGE2 and PGF2 alpha were higher (p < 0.01) in the cancer cells which metastasized to liver as compared with those of the primary intestinal lesion. These results suggest that PGs produced by hepatic metastatic cancer cells might play an important role in cancer metastasis.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Some features of prostaglandin synthesis of the cancer cells metastasized into liver from gastric cancer lesions.

In order to study the mechanism of cancer metastasis, AH100B rat hepatoma cells were transplanted to the stomach of male Donryu rats. Each hepatic metastatic nodule was collected with the respective primary gastric lesions. Each sample thus obtained was injected separately into the peritoneal cavity of male Donryu rats to make free cancer cells; then, intact cancer cells of the hepatic metastatic and primary gastric lesions were collected. After washing in Dulbecco's phosphate-buffered saline (Ca2+ and Mg(2+)-free, pH 7.2), the definite number of the metastatic and primary gastric cancer cells were incubated in the phosphate-buffered saline containing [1-14C] arachidonic acid at 25 degrees C for 30 min. Arachidonic acid metabolites formed during the incubation period were extracted and subjected to thin-layer chromatography, followed by autoradiography. Each radioactive spot was scraped off the plate and measured for its radioactivity. The pattern of the ability to produce PGs was different between the cancer cells which metastasized to the liver and those of the primary lesions, that is, percentage of PGF2 alpha was higher (p < 0.05) and that of PGE2 was quite higher (p < 0.01) in the hepatic metastatic cancer cells as compared with those of the primary gastric lesion. These results suggest that PGs produced by hepatic metastatic cancer cells might play an important role in hepatic metastatic formation.

Animals↗
PubMed1992

Stimulation of arachidonic acid metabolism by a streptococcal preparation (OK-432) in rat peritoneal macrophages.

A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 micrograms/ml). Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.

Animals↗
PubMed1992

Morphological study of amyloid fibrils and preamyloid deposits in the brain with Alzheimer's disease.

In addition to the ultrastructural study of amyloid fibrils, amyloid fibrils and preamyloid in the brain which had the fine ultrastructure of a well-preserved neuropil were examined using methenamine silver stain by light and electron microscope. In serial sections, amyloid fibrils in extracellular spaces continued directly to the capillaries. Using methenamine silver stain, silver granules were deposited at the amyloid fibrils and in extracellular spaces forming diffuse plaques. Many silver granules in the extracellular spaces seemed to strain preamyloid surrounding the capillaries. These findings to indicate that the capillaries have an important role in the formation of amyloid fibrils at least in some senile plaques.

Alzheimer Disease↗
PubMed1992

Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats.

In vivo effects of cytokine-induced neutrophil chemotactic factor (CINC) derived from rats on neutrophil infiltration were investigated using an air-pouch-type inflammation model in rats, and effects of dexamethasone on neutrophil infiltration induced by CINC was also examined in order to gain further insight into the mechanism of antiinflammatory activity of glucocorticoids. Injection of CINC into the air pouch made on the dorsum of rats induced a marked infiltration of neutrophils into the pouch fluid but not mononuclear cells and eosinophils during a 30-min interval after the injection. Maximum effect was induced at a dose of 1.4 micrograms/pouch. Treatment with dexamethasone 3 h before the injection of CINC suppressed the neutrophil infiltration in a dose-dependent manner, but no complete inhibition was observed. CINC injection into the air pouch of rats that had been sacrificed by bleeding in order to minimize neutrophil infiltration from blood stream also stimulated neutrophil infiltration into the pouch fluid when the carcass was incubated at 37 degrees C for 30 min, but the number of infiltrated neutrophils was about 35% of CINC-induced neutrophil infiltration in intact rats. CINC-induced neutrophil infiltration in the carcass, which is supposed to be a reflection of neutrophil migration from extravascular space in subcutaneous tissues to pouch fluid, was not inhibited by dexamethasone treatment. Therefore, the inhibition of neutrophil infiltration by dexamethasone might be due to inhibition of the extravasation of peripheral neutrophils but not due to inhibition of neutrophil chemotaxis from subcutaneous extravascular space to pouch fluid. These findings suggest that clinical effects of steroidal antiinflammatory drugs on neutrophil infiltration in inflammatory disease is partly due to inhibition of neutrophil extravasation induced by preformed neutrophil chemotactic factors in the inflammatory site.

Animals↗
PubMed1992

The distribution of alpha 1-antichymotrypsin and amyloid production in the brain in Alzheimer's disease.

In this immunohistopathological study alpha 1-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer's disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-alpha 1-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, alpha 1-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Analysis of the leukotriene D4 receptor in the granulation tissue of allergic inflammation in rats.

Leukotriene (LT) D4 receptor in the granulation tissue formed in the air pouch-type allergic inflammation model in rats was analyzed. Membrane preparation of the granulation tissue obtained 3-9 days after the antigen challenge has specific binding sites of [3H]LTD4. Scatchard analysis showed that the affinity (Kd) and the density (Bmax) were not changed among the granulation tissue obtained 3-9 days after the antigen challenge. The Kd value in the granulation tissue (0.90 +/- 0.12 nM) was close to that in the rat lung (1.00 +/- 0.24 nM) and the guinea pig lung (0.86 nM). On the other hand, Bmax (62 +/- 8 fmol/mg protein) in the granulation tissue was higher than that in the rat lung (21 +/- 4 fmol/mg protein) but was far less than that in the guinea pig lung (405 fmol/mg protein). LTC4 and LTE4 inhibited the binding of [3H]LTD4 to the membrane preparation of the granulation tissue in a concentration-dependent manner. IC50 of LTC4 and LTE4 were 1 x 10(-7) and 2 x 10(-7) M, respectively. A guanine nucleotide, guanyl-5'-yl-imido-diphosphate (GppNHp), reduced [3H]LTD4 binding to the membrane preparation of the granulation tissue suggesting that LTD4 receptors in the granulation tissue are associated with G proteins. These results indicate that LTD4 binding sites in the granulation tissue are high affinity receptors for LTD4. A possible role of LTD4 in the recurrence of allergic inflammation in the chronic phase is discussed.

Animals↗
PubMed1991

Downward regulation of neutrophil infiltration by endogenous histamine without affecting vascular permeability responses in air-pouch-type carrageenin inflammation in rats.

The role of histamine in neutrophil infiltration and vascular permeability response in carrageenin air pouch inflammation in rats was examined. Injection of carrageenin solution into an air pouch induced a gradual increase in histamine content in the pouch fluid and histidine decarboxylase activity of pouch wall tissues, with a maximum attained at 24 h. Local administration of the H2 antagonists cimetidine and famotidine, but not the H1 antagonist pyrilamine, induced an increase in neutrophil infiltration at 24 h. Both types of histamine antagonists failed to suppress the vascular permeability response. In addition, H2 antagonists attenuated the inhibitory effect of indomethacin on neutrophil infiltration without affecting the indomethacin-induced suppression of vascular permeability response. These results suggest that histamine produced in the inflammatory locus exerts a downward regulation of neutrophil infiltration through H2 receptors but does not play any significant role in the vascular permeability response. Furthermore, the inhibition by indomethacin of neutrophil infiltration might be ascribed to the increase in histamine level in the pouch fluid.

Anaphylaxis↗
PubMed1991

Stimulation of neutrophil adherence to vascular endothelial cells by histamine and thrombin and its inhibition by PAF antagonists and dexamethasone.

1. In order to clarify the roles of platelet-activating factor (PAF) in histamine- and thrombin-induced neutrophil adhesion to vascular endothelial cells, the effects of several PAF antagonists were examined. The effects of the glucocorticoid dexamethasone were also examined in order to gain further insight into the anti-inflammatory actions of glucocorticoids. 2. In culture, histamine and thrombin stimulated the adherence of rat peritoneal neutrophils to human endothelial cells from the umbilical vein. They did not stimulate neutrophil adherence in the absence of endothelial cells, suggesting that the target cells for the histamine- and thrombin-induced adherence of neutrophils were endothelial cells, not neutrophils. 3. Several PAF antagonists, such as CV-3988, L-652,731 and Y-24,180 inhibited the histamine- and thrombin-induced neutrophil adherence in a concentration-dependent manner. Indomethacin failed to inhibit it. 4. Dexamethasone, a steroidal anti-inflammatory drug, did not inhibit the histamine- and thrombin-induced adherence of neutrophils to endothelial cells when the drug was present only during the 20 min incubation period for the adherence assay. When the endothelial cells were preincubated for 3 h with dexamethasone, the adherence of neutrophils to endothelial cells induced by histamine or thrombin was not inhibited. 5. When the neutrophils were preincubated for 3 h with dexamethasone, the histamine- and thrombin-induced adherence of neutrophils to endothelial cells was inhibited. 6. Our studies indicate that: (a) adherence of neutrophils to endothelial cells induced by histamine and thrombin is mediated by PAF production since PAF antagonists inhibited the adherence of neutrophils; and (b) neutrophils, not endothelial cells, are the target cells through which dexamethasone acts to inhibit adherence.

Animals↗
PubMed1991

Characterization of methylated bovine serum albumin-induced allergic inflammation in rats.

An air pouch type allergic inflammation in rats was induced using an insoluble cationic protein, methylated bovine serum albumin (MeBSA), as an antigen. Changes in vascular permeability, local tissue edema, histamine contents in the pouch fluid, and number of infiltrated leukocytes and chemotactic activity in the pouch fluid were analyzed during an 8-hour period after injecting the antigen solution into the air pouch of the immunized and nonimmunized rats. Vascular permeability during the first 30-min interval in the immunized rats was higher than that in the nonimmunized rats, reflecting a higher histamine level in the pouch fluid. However, both the increase in vascular permeability and histamine level in the immunized rats in this period were much lower than those induced by a soluble, noncationic antigen, azobenzenearsonate-conjugated acetyl bovine serum albumin. In the MeBSA-induced allergic inflammation model, a second peak of vascular permeability was induced at 2 h, and local tissue edema formation became apparent at 2 h, reaching a plateau at 4 h. A prominent increase in leukocyte infiltration, especially neutrophils, into the pouch fluid was induced at 4 h in accordance with an increase in chemotactic activity in the pouch fluid. These observations indicate that the acute phase of MeBSA-induced allergic inflammation is characterized by a weak anaphylactic response and a prominent neutrophil infiltration.

Animals↗
PubMed1991

A role of peripheral leukocytes in vascular permeability and edema formation in air pouch type allergic inflammation in rats.

Using an air pouch type allergic inflammation model in rats, a role of circulating leukocytes in allergic inflammation responses was investigated by comparing normal rats with peripheral leukocytopenia rats induced by cyclophosphamide treatment. In the leukocytopenia rats, vascular permeability, edema formation, leukocyte infiltration into the pouch fluid, neutrophil chemotactic activity in the pouch fluid occurred both at the early phase (4 h) and the late phase (8 h) after the antigen challenge were decreased. However, edema formation induced by intradermal injection of arachidonate metabolites, serotonin, or platelet activating factor was not suppressed at all in the leukocytopenia rats. A possible role of peripheral leukocytes in allergic and non-allergic inflammation is discussed.

Animals↗
PubMed1991

A difference in prostaglandin-producing ability between cancer cells metastasized into liver and kidney.

In order to study the mechanism of cancer metastasis AH100B cells, a rat hepatoma cell line, were injected into the left carotid artery of male Donryu rats to form metastatic lesions. Each metastatic nodule in the liver and kidney was collected and injected into the peritoneal cavity of normal rats. About 3 weeks later, intact metastatic cancer cells were collected from each ascites that was not bloody. After washing in Dulbecco's phosphate-buffered saline (PBS, Ca2+ and Mg(2+)-free, pH 7.2), 1 x 10(6) cancer cells were incubated in the PBS containing [1-14C]-arachidonic acid (AA) at 24 degrees C for 5 min. AA metabolites formed during the incubation period were extracted and subjected to thin layer chromatography, followed by autoradiography. Each radioactive spot was scraped off the plate and its radioactivity was measured. In the cancer cells which metastasized to the liver, the ability to produce prostaglandin (PG) E2 was higher (p less than 0.05) but those to produce PGF2 alpha and 6-keto-PGF1 alpha were lower (p less than 0.01) than in the cancer cells which metastasized to the kidney. These results suggest that cancer cells metastasizing to the liver and the kidney are different from each other in the ability to produce PG.

Animals↗
PubMed1991

A transgenic mouse line developed to express human amyloidogenic transthyretin cDNA in the brain.

To elucidate the role of amyloid deposits in the pathogenesis of degenerative disorders of brain, we attempted to develop transgenic mice producing amyloidogenic human variant transthyretin (TTR) in the brain. A minigene, in which the promoter region of the mouse myelin basic protein (MBP) gene was ligated to the human mutant TTR cDNA, was used for microinjection. A transgenic mouse line expressing the human TTR mRNA specifically in the brain was thus derived. However, human TTR was not detected in the brain or in the serum of these mice. These data suggest that the human mutant TTR mRNA is not efficiently translated in the mouse brain.

Amino Acid Sequence↗
PubMed1990

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages.

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.

Alkaloids↗