We examined the effect of murine kidney extract (MKE) on the clonal growth of mast cells from murine peritoneal cells. Adding MKE resulted in a 40% inhibition of colony formation of mast cells in a methylcellulose culture, and a 90% decrease in mast cell numbers and histamine content in mast cells in a liquid culture containing stem cell factor and interleukin-3. The mast cell inhibitory factors in MKE were heat sensitive proteins of approximately 560 and 24 kDa. These results suggest that MKE contains regulators that suppress the growth of murine mast cells and histamine synthesis.

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We have previously reported the isolation of human gelatin-binding protein 28 (GBP28) gene which is specifically expressed in adipose tissue. The transcriptional activity of the flanking region of the GBP28 gene was examined by the transient transfection of promoter-luciferase reporter constructs into 3T3 adipocytes and electrophoretic mobility shift assay. This revealed the existence of a protein which binds to the 5'-flanking region of the GBP28 gene in nuclear extracts from human adipose tissue, but not in nuclear extracts from mouse liver. The C/EBP sites contained in this region are thought to take part in the regulation of GBP28 gene expression.

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We demonstrate that glycyrrhizin (GL) enhanced Fas-mediated apoptotic body formation and DNA fragmentation in T cell lines although GL alone did not induce apoptosis. The enhancement effect of Fas-mediated apoptosis by GL was dose-dependent above 0.3 μM. Time course study revealed that simultaneous co-treatment of GL and anti-Fas antibody was crucial for the enhancement of apoptosis and pretreatment with GL was not effective. Anti-Fas antibody elicited caspase-3-like activity. However caspase-3-like activity with co-treatment of GL and anti-Fas antibody was the same level as the antibody alone. Glycyrrhetic acid, the aglycon of GL, did not enhance Fas-mediated apoptosis. The amphipathic property of GL might enable it to interact with the plasma membrance and lead to the enhancement of apoptosis.

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Lactoferrin (LFR) plays an important role in the anti-microbial defense through iron binding, lipopolysaccharide binding and immunomodulation. In this study, we demonstrate that bovine LFR specifically inhibits the hemolytic activity of listeriolysin O (LLO) produced by Listeria monocytogenes. The hemolytic activity of LLO was completely inhibited in the presence of bovine LFR that was highly purified on two cation-exchange columns, whereas that of streptolysin O or perfringolysin O was not inhibited at all. A rabbit anti-LFR antibody canceled this inhibitory activity of bovine LFR. Although human transferrin exhibits 62% amino acid identity with bovine LFR, human apo-transferrin could not inhibit LLO-induced hemolysis. An increase in the concentration of FeCl₃ or the Fe³⁺-saturation of bovine LFR, however, slightly reduced its inhibition of the hemolysis.The inhibitory activity of bovine LFR was dependent on pH, since it was observed under neutral and alkali conditions, but not under acidic conditions. These results suggest that the inhibition of LLO-induced hemolysis by bovine LFR is influenced by pH and iron ions, both of which may lead to conformational changes of LFR.

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In the present research we examined the levels and types of arginine amidase activities that were released from isolated rabbit arteries treated with heparin or chondroitin sulfate. Heparin accelerated the release of arginine amidase activity from the isolated rabbit ear artery, the induction was not significant; a slight increase in activity was observed in the level of arginine amidase released from isolated rabbit aorta, but no significant difference was observed. On the other hand, it was revealed that the addition of chondroitin sulfate, accelerated this release from isolated rabbit ear artery with 5% significant differences.After the addition of chondroitin sulfate, the arginine amidase activity released from isolated rabbit arteries was analyzed using various affinity adsorption methods. This analysis confirmed the presence of two types of fibrinolytic enzymes : plasminogen/plasmin activity and plasminogen activators, but no thrombin was detected.

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To explore the role of glutathione as a neuromodulator, we investigated effects of reduced (GSH) and oxidized glutathione (GSSG) on drug-induced convulsions in mice. Intracerebroventricular administration of GSH or GSSG (10-300 nmol) did not produce convulsions. When GSH was administered prior to subcutaneous administration of pentylenetetrazol (80 mg/kg), it significantly inhibited pentylenetetrazol-induced convulsions. The inhibitory effect of GSH was dose dependent, and mimicked by GSSG. In addition, neither GSH nor GSSG affected convulsions induced by subcutaneous administration of N-methyl-DL-aspartate (400 mg/kg). These findings suggest that glutathione has a specific anticonvulsive effect.

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We examined the effects of Bu-Zhong-Yi-Qi-Tang (Japanese name : Hochu-ekki-to, HET), a traditional Chinese medicine, on IgE production and histamine release in mice immunized intraperitoneally with a mixture of ovalbumin (OA) and aluminum hydroxide (alum adjuvant). Three groups of mice were orally administered 0, 1.7 or 17 mg of HET on day 13 after the first immunization with a mixture of 1 μg OA and 1 mg alum adjuvant. They were again immunized with the same dose of OA plus alum adjuvant on day 14. The immunological changes in mice treated with OA alone or OA plus HET were examined, and the following findings were obtained. In the HET-treated mice, the elevation of anti-OA IgE in serum, and histamine release from basophils in blood, were significantly suppressed. A significant suppression of interleukin-4 (IL-4) secretion and proliferation of splenic lymphocytes in primary culture was also observed. A tendency to suppress the elevation of anti-OA IgG1 in serum and interleukin-2 (IL-2) secretion from splenic lymphocytes was observed in the HET-treated mice. These findings suggest that oral administration of HET suppresses IgE antibody production and histamine release in type I allergic reaction in mice immunized with OA plus alum adjuvant; this shows the efficacy of HET in treating type I allergic diseases, such as asthma.

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We previously found that 72-kDa heat shock protein (Hsp72) was induced and accumulated in the nuclei, together with DNA damage, in human alveolar epithelial (L-132) cells by exposure to dimethylarsinic acid (DMAA), which is a main metabolite of inorganic arsenics in mammals. In the present study, the intracellular behavior of Hsp72 was investigated during the recovery from the DNA damage induced by exposure to DMAA. L-132 cells were exposed to 10 mM DMAA for 3 h, and then incubated in DMAA-free medium. The induction of Hsp72 by exosure to DMAA reached a peak at 6-9 h after removal of DMAA. However, the cell-nuclear distribution of Hsp72 was observed until 3 h after the start of DMAA-free incubation. We further investigated the appearance of apoptosis of L-132 cells after exposure to 10 mM DMAA for 3 h. Internucleosomal DNA fragmentation and morphological changes, as criteria for the evidence of apoptosis, were observed 6-22 h after the start of DMAA-free incubation. The appearance of apoptosis was followed by the release of Hsp72 from the cell nuclei. These results suggest a possibility that the cell-nuclear Hsp72 may suppress the appearance of apoptosis in DNA-damaged cells.

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Fractionation of the 50% ethanol extract of Polyporus umbellatus Fries by column chromatography on Amberlite XAD-2, silica gel, Sephadex LH-20 and octadecyl silica gel (ODS) (C18) monitored by a hair-regrowth activity assay, afforded three active principles, 1, 2 and 3. The structures of 1, 2 and 3 were determined as acetosyringone, polyporusterone A, and polyporusterone B by comparison of their spectral data with that of authentic samples, respectively. The effects of several compounds related to acetosyringone, 3, 4-dihydroxybenzaldehyde or polyporusterone A on hair regrowth were also investigated.

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Three new 6-O-acylated isoflavone glycosides were isolated from soybeans fermented with Bacillus subtilis (natto) and identified as daidzein 7-O-β-(6"-O-succinyl)-D-glucoside (1), genistein 7-O-β-(6"-O-succinyl)-D-glucoside (2), and glycitein 7-O-β-(6"-O-succinyl)-D-glucoside (3) on the basis of spectral data and chemical transformations. During fermentation, the content of the isoflavone glycosides first decreased and then increased, whereas the corresponding 6"-O-succinyl derivatives first accumulated and then decreased, in either soybeans or soybean cooking solution. These changes suggest that enzymatic interconversion of isoflavone glycosides and the corresponding 6"-O-succinylated derivatives occurs in these media during fermentation.The 6-O-succinylated isoflavone glycosides 1, 2 and 3 accounted for 4.8, 7.2 and 0.6%, respectively, of the total isoflavones in commercial fermented soybeans (Japanese natto).Oral administration of 1 or 2 alone for 4 weeks at a dose of 50 mg/kg/d prevented bone loss in ovariectomized (ovx) rats fed a calcium-deficient diet, being as effective as the positive controls, daidzin and genistin, respectively. Compound 1 seems to be proestrogenic, like daidzin, which suppresses bone resorption to prevent bone loss after ovariectomy by directly acting on bone sites, while 2 appears to have a different mechanism of action, like that of genistin.

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The photoproducts produced by irradiating 8-methoxypsoralen (8-MOP) in the presence of spermine (Spm) were fractionated using gel filtration chromatography (GFC) on a Sephadex G-25 column. As a result, two bands which were characterized by the effects on hyaluronidase activity were obtained. The first band strongly activated the hyaluronidase, but a second band did not exhibit any effect on the enzyme activity. The first and second bands contained photoproducts with molecular weights (MW)>2700 and MW<728, respectively, determined by the GFC method. The photoproducts, 8-MOP-Spm-P_GFC obtained from the first band, but not the photoproducts with lower MW from the second band, showed enzyme activating action.8-MOP-Spm-P_GFC induced paw edema, which was stronger in the first phase than the second one in rats, differing from that induced by carrageenin. This photoproduct was a substance with lower cell toxicity because it did not cause hemolysis on red blood cells or the release of lactic dehydrogenase from mast cells in rats.The effects of various drugs on 8-MOP-Spm-P_GFC-induced edema were investigated. As a results, edema formation was inhibited by drugs with an anti-histaminic action, such as alimemazine, dl-chlorpheniramine, promethazine, ketotifen and azelastine, and with anti-serotonin action such as cyproheptadine. On the other hand, tranilast did not show significant inhibition and indomethacin showed a tendency to increase its formation.These results suggested that 8-MOP-Spm-P_GFC is a new inflammatory substance and is very useful as an agent to develop new anti-inflammatory drugs without cyclooxygenase inhibitory action.

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The time-dependent inactivation of aromatase by epoxy analogs of the good aromatase inhibitors, androst-4-ene-6, 17-dione (3) and androst-5-ene-4, 17-dione (7), 4β, 5β-epoxy and 5β, 6β-epoxy compounds 10 and 13 and their 19-oxo derivatives 11 and 14, was examined in either the presence or absence of NADPH. The 4β, 5β-epoxy-19-oxo steroid 11 along with the 19-methyl-5β, 6β-epoxide 13 inactivated human placental aromatase in a mechanism-based manner, in the presence of NADPH, with rate constants for inactivation (k_inact) of 0.133 min^-1 for steroid 11 and 0.100 min^-1 for steroid 13, whereas the two other steroids, 10 and 14, did not. On the other hand, none of four epoxides studied caused time-dependent inactivation of aromatase in an affinity-labeling manner in the absence of NADPH. These results are the first report showing that inhibitors 11 and 13 are suicide substrates having an epoxyketone structural featue.

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We have previously proposed a novel deconvolution method, which can estimate first-pass metabolism of orally administered drugs. In the present study, we examined whether this deconvolution method is useful for evaluating oral dosage forms. The absorption and first-pass metabolism of orally administered aspirin formulated in several forms were analyzed. Two types of microcapsules consisting of Eudragit L100 alone and Eudragit L100/ethylcellulose (4 : 6) were prepared as sustained release formulations, for comparison with aspirin in powder form. The deconvolution analysis revealed that absorption of aspirin was sustained by encapsulating it in microcapsules. Interestingly, it also revealed that the percentage metabolized during abosrption was different among the three types of formulations. Thus, the deconvolution method has enabled a comprehensive analysis of orally administered drugs. This method is believed to contribute to the evaluation of oral drug formulations.

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The effect of naloxone, a potent and specific opioid antagonist, on cerebral glucose utilization was investigated in conscious rat. For quantitative evaluation of the functional activity in brain, the regional cerebral metabolic rate for glucose (rCMR_glc) was measured by the double tracer technique, using [¹⁴C]2-deoxyglucose and [³H]3-O-methylglucose. Intravenous administration of naloxone significantly increased rCMR_glc in the medulla and thalamus at a dose of 1 or 10 mg/kg, and in the cerebral cortex, midbrain and cerebellum at a dose of 10 mg/kg. Our findings strongly suggest that naloxone by itself affects the cerebral functional activity within 10 min of administration.

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To establish a speedy preparation method for the fibrinogen-rich fraction (FRF) from autologous plasma using fibrin glue, we compared the concentrations and yields of coagulation factors in FRF prepared by 3 methods. Human plasma from healthy volunteers was divided into 3 samples. Two samples were frozen at -20°C in a freezer and defrosted in a 4°C water bath. One sample of defrosted plasma was centrifuged and FRF was obtained (C method). Another sample of defrosted plasma was filtered and FRF was obtained (F method). The last sample was treated with cold ethanol(1/10) in a 4°C water bath and FRF was obtained after centrifugation (E method). The concentrations of fibrinogen, fibronectin, factor XIII, and plasminogen in each obtained FRF were measured and yields were calculated. (1) The volume of FRF obtained by the E method was greater than that by the C method, but less than that by the F method. While the variation in volume obtained by the E method was the lowest among the 3 methods; (2) the concentrations of fibrinogen obtained by the E and C method were similar, but the yield from the E method was the highest; (3) the concentration and yield of fibronectin from the E and C method were similar and were greater than those by the F method; (4) the concentration and yield of factor XIII from the E method were significantly higher than those from the other methods; (5) the E method preparation time was about 1 h, the shortest among the 3 methods. These results indicate that high quality FRF from autologous plasma can be prepared easily and within 1 h by the E method.

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Protective effect of the cellular ubiquinone (UQ) reducing system linked to cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) against hydrogen peroxide (H₂O₂)-induced lipid peroxidation was investigated using UQ and control hepatocytes freshly isolated from rats injected with UQ-10 and the vehicles 14 d in advance, respectively. The UQ hepatocytes had higher levels of ubiquinol (UQH₂)-10 content and NADPH-UQ reductase activity than the control hepatocytes but did not differ in other antioxidant factors from the latter cells. The UQ hepatocytes exhibited higher cell viability and lower release of lactate dehydrogenase than the control hepatocytes when they were exposed to H₂O₂ of up to 100 mM for 1 h at 37°C. Furthermore, the formation of thiobarbituric acid reactive substances (TBARS) by H₂O₂ was almost completely inhibited in the UQ hepatocytes. Decreases in UQH₂ and α-tocopherol contents and NADPH-UQ reductase activity by H₂O₂ exposure were observed in both types of the hepatocytes, but those levels in the UQ hepatocytes after the exposure were still higher than in the control hepatocytes. The decreases in ascorbic acid, reduced glutathione and protein thiol contents and DT-diaphorase activity by H₂O₂ were not different between in the two types of hepatocytes. Antioxidant enzyme activities of catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione reductase in the hepatocytes were not inhibited by H₂O₂. From these results, it was concluded that the cellular UQ reducing system linked to cytosolic NADPH-UQ reductase functions mainly as an antioxidant defense for cellular membranes.

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Five 2-pyridinecarboxylic acid-related compounds (1, 2 and 5-7) showed germination inhibition against the seed of Brassica campestris L. subsp. rapa HOOK fil et ANDERS at a concentration of 5.0×10^-4M. These compounds also demonstrated inhibitory activity on the growth of the root of this plant at a concentration of 3.0×0^-4M; among these compounds, 2-pyridylacetic acid (5) showed the strongest inhibitory activity, and the effect was slightly stronger than that of sodium 2, 4-dichlorophenoxyacetate (2, 4-D) used as a positive control. The amounts of chlorophyll in the cotyledons of this plant treated with these active compounds was lower than that of the control group. Four compounds (1 and 5-7) with germination inhibition also showed inhibitory activities against α-amylase and carboxypeptidase A, and 5 was the strongest inhibitor toward both enzymes.

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The preventive effect of propolis extract on D-galactosamine-induced hepatic injury was examined in rats. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were significantly increased at 24 h after intraperitoneal injection of D-galactosamine (400 mg/kg) in the animals. Propolis extract was administered orally three times in doses of 3 or 30 mg/kg at 18 h and 1 h before and 8 h after D-galactosamine injection. The extract itself and the vehicle alone (dextran) caused no significant changes in serum AST or ALT activities. Treatment with the extract dose-dependently prevented the increases in serum AST and ALT activities induced by D-galactosamine, and significant inhibition was observed at a dose of 30mg/kg. These results suggested that propolis extract may have an ameliorating effect on hepatic dysfunction.

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Effects of a selective serotonin reuptake inhibitor, zimelidine, on plasma glucose was studied in mice. Zimelidine dose-dependently induced hyperglycemia, although it did not change insulin levels. To determine the involvement of the serotonergic system in zimelidine-induced hyperglycemia, effects of the 5-HT depleter p-chlorophenylalanine(pCPA) were examined. pCPA significantly reduced zimelidine-induced hyperglycemia. This suggests that zimelidine-induced hyperglycemia is mediated by the serotonergic system through its 5-HT reup-take inhibition.

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The sensory neuropeptide substance P is known to be involved in neurogenic inflammation. We examined the effect of substance P on neutrophil adhesion to human umbilical vein endothelial cells (HUVEC). Stimulation of HUVEC with substance P increased their adhesion to neutrophils in a time- and concentration (10-<-10>-10^-7 M)-dependent manner. The adhesion was inhibited by the tachykinin NK₁ receptor antagonist (+)-(2S, 3S)-3-(2-Methoxybenzylamino)-2-phenylpiperidine (CP-99, 994) and also by the protein kinase C inhibitors 1-(5-Isoquinolinesulfonyl)-2-methyl piperazine (H-7) and bisindolylmaleimide (BIM), but not by the protein kinase A inhibitor N-[2-(p-Bromocinnamylamino) ethyl]-5-isoquinoline sulfonamide (H-89). These results indicate that substance P induces adhesion of neutrophils to HUVEC by activation of protein kinase C via the NK₁ receptor on the HUVEC.

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Aldehyde oxidase was purified from hamster liver cytosol by ammonium sulfate fractionation, chromatography on DEAE-cellulose and Phenyl-Toyopearl, and HPLC-gel filtration on TSK-gel G3000SW_XL column. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was determined to be 144800 by SDS-PAGE and 288000 by HPLC gel filtration. The isoelectric point was pH 5.1. The apparent K_m and V_max for benzaldehyde and 2-hydroxypyrimidine were 19.0 and 4.4μM, and 165 and 211 nmol/min/mg protein, respectively. The benzaldehyde oxidase activity was markedly inhibited by menadione and chlorpromazine. The substrate specificity was different from those of the enzymes from other animals.

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Four active principles, 1, 2, 3 and 4, were isolated from Polygara senega var. latifolia TORR. et GRAY by a combination of partition and column chromatography on silica gel and octadecyl silica gel (ODS), monitored by a hair-regrowth activity assay. Compounds 1, 2, 3 and 4 were identified as senegose A, senegin II, senegin III, and senegasaponin b by comparison of their spectral data with those of authentic samples.

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The antitumour activity of the methanolic extract of Glinus lotoides (MGL) has been evaluated against Dalton's ascitic lymphoma (DAL) in Swiss albino mice. A significant enhancement of mean survival time of tumour bearing mice and peritoneal cell count in normal mice was observed with respect to the control group. When these MGL treated animals underwent i.p. inoculation with DAL cells, tumour cell growth was found to be inhibited. After 14 d of inoculation, MGL is able to reverse the changes in the haemotological parameters, protein and packed cellular volume consequent to tumour inovulation.

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The purpose of this study was to characterize the ocular pharmacokinetics of a beta-blocker, tilisolol, after instillation into anesthetized rabbits using a mathematical model including a diffusion process. The samples were analyzed by HPLC. Anesthetized rabbit was used as a model of tear secretion deficiency. Anesthetized rabbits showed higher drug concentration in the tear fluid and aqueous humor after instillation than unanesthetized rabbits. A mathematical model including a diffusion process and in vivo penetration parameters well described the concentrations of tilisolol in the aqueous humor after instillation in anesthetized rabbits.

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