We have investigated the effect of inhibitors of glycoprotein processing on cytokine secretion and production in anti CD3-stimulated T cells to elucidate the role of carbohydrate in the triggering of T cell function. The inhibitors of glycoprotein processing, especially mannnosidase inhibitors, enhanced the anti CD3-induced production of interleukin-2 (IL-2), which is a cytokine without the linkage sequence of N-linked oligosaccharides. On the other hand, N-methyl-1-deoxynojirimycin (NMdNM, an inhibitor of processing glucosidase I), 1-deoxynojirimycin (dNM, an inhibitor of processing glucosidase I and II) and bromoconduritol (BCD, an inhibitor of processing glucosidase II) inhibited the secretion of interleukin-4 (IL-4), interferon-γ (IFN-γ), or interleukin-5 (IL-5) into culture supernatants of anti CD3-stimulated T cells, which had N-linked oligosaccharides. Mannosidase inhibitors, 1-deoxymannojirimycin (dMAN, and inhibitor of processing mannosidase I) and swainsonine (SWN, an inhibitor of processing mannosidase II) did not inhibit the secretion or production of IL-4, IFN-γ and IL-5. To confirm the inhibition of N-linked oligosaccharide processing in the cytokines by the above inhibitors, the binding of IFN-γ to lectins with various sugar-binding specificities was investigated. All inhibitors reduced the binding of IFN-γ to PHA E4, which had a high affinity to bi- or tri-antennary complex type N-linked oligosaccharides with bisecting N-acetylglucosamine. Similarly, all inhibitors reduced the binding of IFN-γ to PHA L4, which had high affinity to tri- or tetra-antennary complex type N-linked oligosaccharides with β1-6-linked branching. SWN and dMAN increased the binding of IFN-γ to concanavalin A (ConA), which had a high affinity to bi-antennary complex type, hybrid type and high-mannose type N-linked oligosaccharides. These results suggest that the processing inhibitors used here inhibit the N-linked oligosaccharide processing of cytokines, and the inhibition of processing enzyme glucosidases I and II induces a decreased secretion of cytokines with N-linked oligosaccharides.

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Eubacterium sp. GLH with Ruminococcus sp. PO1-3 and Clostridium innocuum ES24-06 possessing enzymes involved in the metabolism of glycyrrhizin (GL) was cultured in GAM medium with and without 1.0 mM GL or 1.0 mM glycyrrhetic acid (GA). GL (1.0 mM) enhanced 3α-hydroxyglycyrrhetinate (3α-hydroxyGA) dehydrogenase activity, GA (1.0 mM) suppressed 3α-hydroxyGA dehydrogenase activity, GL β-D-glucuronidase activity and the mixed bacterial growth, and GL and GA showed almost no change in a lower level of 3β-hydroxysteroid dehydrogenase (3β-HSD) activity during 5 d of culture. GL (1.0 mM) and GA (1.0 mM) were metabolized to a small amount of GA and a negligible amount of 3-oxo-glycyrrhetic acid (3-oxo-GA) and 3α-hydroxyGA, and to a negligible amount of 3-oxo-GA, respectively, by these mixed bacteria. These amounts coincided with those of metabolites produced from 1.0 mM GL and 1.0 mM GA added to these mixed bacteria after 24 h culture. Whole bacteria and sonicated bacteria derived from the collection of these mixed bacteria reached a maximal stage and metabolized GL to a relatively large amount of GA and 3-oxo-GA, and a negligible amount of 3α-hydroxyGA and GA to a small amount of 3-oxo-GA and 3α-hydroxyGA within 180 min. GL β-D-glucuronidase with 3β-HSD and 3α-hydroxyGA dehydrogenase partially purified from each bacterium was converted GL to 3α-hydroxyGA, production metabolites of about 60% after 10 min of incubation. These mixed bacteria possessed high enzyme activities could produce the metabolites of GL in under one hour under conditions.

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A novel assay for a peroxisomal β-oxidation enzyme by sandwich ELISA using a monoclonal antibody (RPX-5) against purified rat liver peroxisomes was developed. Immunoblot analysis revealed that RPX-5 recognized a 78 Kd protein, which is a peroxisomal bifunctional enzyme (PBE) in the β-oxidation pathway. Immunoprecipitation by RPX-5 and the resulting reduction of PBE activity were dependent on RPX-5 concentrations. Sandwich ELISA using RPX-5 could be used to assay PBE in the range of 30 to 2000 ng protein/ml. In rat hepatocyte cultures, the PBE amount by this assay correlated well with PBE activity, with correlation coefficients of 0.965. Studying the mechanisms of peroxisomal induction, patterns of peroxisomal induction were examined by co-treatment of rat hepatocytes with various peroxisome proliferators (PxPs). Treatment with clofibrate and bezafibrate resulted in neither an additive nor synergistic effect on PBE level. On the other hand, co-treatment with either bezafibrate-Wy-14, 643 or clofibrate-MEHP(mono(2-ethylhexyl)phthalate) both resulted in an additive effect. From these results, it is suggested that PxPs of the fibrate group may exert their functions via a common process, and non-fibrate PxPs via a different process in hepatocytes. The cognition site for peroxisome proliferators, therefore, might not involve a single site for inducing peroxisomal enzymes.

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The characteristics of isolated guinea-pig ileal contractions of basal tension after tetanic stimulation in the presence of a high concentration of naloxone (NLX) [post-tetnic contraction] were investigated. The post-tetanic contraction did not occur in the absence of NLX, but did occur in a concentration-dependent manner in the presence of a high concentration of NLX (5×10^-7, 10^-6, 10^-5 and 5×10^-5 M), the concentration of which was higher than that required for antagonizing post-tetanic twitch inhibition. The contraction in the presence of 10^-6 M NLX was diminished by washing NLX from the preparation with Krebs-bicarbonate solution. The contraction under 10^-6 M NLX occurred in a frequency-dependent manner (5, 10 and 20 Hz), but not at 0.1 Hz. Tetanic stimulation (5, 10 and 20 Hz) without NLX did not induce this contraction. The post-tetanic contraction with 10^-6 M NLX had a tendency to be antagonized in the presence of 5×10^-6 M atropine. Methysergide (5×10^-5 M) had no effect on this contraction. Spantide (10^-5 M) largely inhibited the contraction, and indomethacin (5×10^-6 M) and tetrodotoxin (5×10^-7 M) completely inhibited this contraction. These results indicate that tetanic stimulation in the presence of a high concentration of NLX induces contraction of the ileal muscle due to the release of endogenous ileal contractile substances (substance P, prostalandins and acetylcholine), and suggests that these contractions are closely linked to the endogenous opioid system induced by tetanic stimulation in the ileum.

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Antidiabetic effects of white skinned sweet potato (Ipomoea batatas L.) (WSSP) and troglitazone, an insulin sensitizer, were investigated. Hyperinsulinemia in Zucker fatty rats was reduced by 23%, 26%, 60% and 50%, respectively, 3, 4, 6 and 8 weeks after starting the oral administration of WSSP. Similar results were obtained with troglitazone. In the glucose tolerance test after 7 weeks of treatment, increases in blood glucose levels after glucose loading were inhibited by the administration of WSSP. Glucose tolerance was also improved. Blood triacylglyceride (TG) and free fatty acid (FFA) lactate levels were lowered by the oral administration of WSSP. Similar effects on blood insulin, lipid and lactate levels were observed after the administration of troglitazone. Body weight gain increased in the troglitazone group, but not in the WSSP group, compared to the control group. In histological examinations of the pancreas of Zucker fatty rats, remarkable regranulation of pancreatic islet β-cells was observed in the WSSP and troglitazone groups after 8 weeks of treatment. These results suggest that WSSP shows remarkable antidiabetic activity and improves the abnormality of glucose and lipid metabolism by reducing insulin resistance.

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The three casein kinase II (CK-II) phosphate acceptors (p35, p17 and p15) in the Superdex CK-II fraction prepared from a 1.5 M NaCl extract of porcine liver were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC) as a heterocomplex associated with CK-II. Determination of the N-terminal amino acid sequences and immunological tests confirmed that these three CK-II phosphate acceptors belong to the family of 60S acidic ribosomal proteins (P0, P1 and P2). Three polyphenol-containing anti-oxidant compounds [catechin, epigallocatechin gallate (EGCG) and quercetin] inhibited CK-II activity (phosphorylation of these ribosomal P proteins) in a dose-dependent manner in vitro. Quercetin (ID₅₀n=approx. 50 nM) was found to be an effective CK-II inhibitor. In contrast, CK-II activity was significantly stimulated by lower doses (0.3-3 μM) of GL, but was inhibited at high doses above 30 μM. As expected, GL at high doses above 200 μM inhibited the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera from patients with systemic lupus erythematosus (SLE). These results suggest that (i) a GL-affinity column is useful for effective purification of 60S acidic ribosomal P proteins from various mammalian cells as a heterocomplex associated with CK-II; and (ii) a relative high dose of GL may prevent the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera of SLE patients.

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The amelioration of secretory diarrhea has been reported after the administration of zaldaride maleate (ZAL), a selective calmodulin inhibitor, to male rodents. In this study, the antidiarrheal effect of ZAL in female rats was compared with that in male rats. In female and male rats, ZAL significantly ameliorated 16, 16-dimethyl prostaglandin E₂-induced diarrhea at doses of 1 and 3 mg/kg (p.o.), respectively, with ID₅₀ values of 0.7 mg/kg (p.o.) in the females and 10.3 mg/kg (p.o.) in males. In castor oil-induced diarrhea, ZAL also significantly reduced the incidence of diarrhea in female and male rats at doses of 10 and 30 mg/kg (p.o.), respectively, When the same dose of ZAL was given orally to female and male rats, the maximum plasma level of this compound was approximately 3 times higher in female rats than in male rats. In contrast, after intravenous administration of the same dose of ZAL to female and male rats, the total clearance of this compound was similar. In an Ussing chamber experiment, the inhibitory action of ZAL on vasoactive intestinal polypeptide-induced ion secretion in the colon showed no difference between female and male rats. In conclusion, the antidiarrheal effect of ZAL in female rats is more potent than that in males, and could be due to the difference in plasma levels of this compound between female and male rats after oral administration.

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The influence of oxidative stress by hydrogen peroxide (H₂O₂) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30min in saline A containing H₂O₂. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H₂O₂. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addtion of antioxidants (N-acetylcysteine, L-ascorbic aicd), a metal-chelator (1, 10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H₂O₂-induced cell viability reduction suggested that oxidative stress by H₂O₂ itself or H₂O₂-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.

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The formation of 7-oxo-Δ⁸-tetrahydrocannabinol (7-oxo-Δ⁸-THC) from 7β-hydroxy-Δ⁸-THC was found in hepatic microsomes of rats. The activity was stereoselective and about 3-fold higher than that from 7α-hydroxy-Δ⁸-THC. The oxidative activity of 7α- and 7β-hydroxy-Δ⁸-THC to 7-oxo-Δ⁸-THC was significantly higher in male than in female, and significantly enhanced by both dexamethasone and phenobarbital, and then inhibited up to about 20% of the control value by antibody against P450GPF-B, presumably a member of the 3A subfamily, a major enzyme responsible for the formation of 7-oxo-Δ⁸-THC in guinea pigs. This antibody also inhibited the formation of 7α- and 7β-hydroxy-Δ⁸-THC, and 7-oxo-Δ⁸-THC from Δ⁸-THC by hepatic microsomes of rats. These results indicate that there is a sex-related difference in the oxidatio of 7-hydroxy-Δ⁸-THC to 7-oxo-Δ⁸-THC and the reaction is mainly catalyzed by P450 enzyme(s) beloging to the 3A subfamily as major enzyme(s) of microsomal alcohol oxygenase in rats.

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Seven flavonoids of Stachys chrysantha and Stachys candida have been isolated. The structures of the compounds were elucidated by spectroscopic methods, particularly highfield NMR spectroscopy. The effects of the methanol extracts of these two endemic Greek Stachys sp. and their main flavonoids were examined on arachidonic acid (AA) metabolism in the cellular system (mouse peritoneal macrophages and human platelets). Their cytotoxicity on cells was also investigated. Most samples assayed did not exhibit any significant effect on prostaglandin E₂ (PGE₂)-release from calcium ionophore-stimulated mouse peritoneal macrophages. Only chrysoeriol-7-O-β-D-(3"-E-p-coumaroyl)-glucopyranoside, at the highest non-cytotoxic dose (50 μM), inhibited the release of PGE₂, but this effect is not statistically significant. The release of leukotriene C₄ (LTC₄) by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by a crude extract of S. chrysantha, with an IC₅₀ value of 34.3 μg/ml. Xanthomicrol (IC₅₀=29.2 μM) and chrysoeriol-7-O-β-D-(3"-E-p-coumaroyl)-glucopyranoside (IC₅₀=11.1 μM) also inhibited the release of LTC₄, althouth it showed less potency than the reference compound nordihydroguaiaretic acid (NDGA) (IC₅₀=2 μM). However, most samples assayed showed a significant effect on thromboxane B₂ (TXB₂)-release from calcium ionophore-stimulated human platelets, with inhibition percentages slightly lower than the reefrence drug ibuprofen (IC₅₀=7 μM). The IC₅₀ values are : crude extract of S. candida 23.3 μg/ml; crude extract of S. chrysantha 23.1 μg/ml; xanthomicrol 28.8 μM; calcycopterin 2.66 μM and chrysoeriol-7-O-β-D-(3"-E-p-coumaroyl)-glucopyranoside 8.8 μM. Our results indicate that the selective inhibition of TX-synthase enzyme may be the primary target of action of most of these samples, and one of the mechanisms through which thus exert their antiinflammatory effects.

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The effect of a methanol extract of Eucommiae Cortex on collagen synthesis was investigated in false aged model rats. Granuloma formation and collagen snthesis were significantly increased by the administration of the methanol extract of Eucommiae Cortex. The effective component of Eucommiae Cortex was then discussed by fractionating the methanol extract of Eucommiae Cortex. Eucommiol, a main component in the water fraction of the methanol extract, was found to be an effective compound. In our previous paper, we reported the promoting effect of Eucommia ulmoides OLIVER leaf on collagen synthesis, and found geniposidic acid and aucubin were the main effective compounds in the leaf. Based on our data in this paper, we clarified that the main effective components of the Eucommia ulmoides OLIVER leaf and Eucommiae Cortex were different. Geniposidic acid and aucubin were reported to be contained at a high concentration in the fresh cortex of Eucommia ulmoides OLIVER, but during the drying process and storage, most of them were destroyed by enzymes in the cortex and very little remained in the Eucommiae Cortex. Therefore, we investigated the effect of the methanol extract of fresh cortex of Eucommia ulmoides OLIVER. A stronger effect than Eucommiae cortex was shown, and geniposidic acid, aucubin and geniposide were concluded to be the main effective components. Although geniposide was found to be an effective compound, when the dose was higher than 50 mg/kg/d, toxicity was shown. The pharmaceutical effect of eucommiol was reported for the first time.

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We have reported that collagen metabolism was improved by the administration of Eucommia ulmoides OLIVER leaf. In this paper, we examine the granuloma maturation and deposition of collagen in the granuloma of rats due to the oral administration of this leaf. After 3 weeks of the oral administration, granuloma formation was induced by the formalin soaked filter paper-pellet method. A week later, the developing granuloma was dissected. Granuloma formation was significantly increased due to ingestion of the dried leaf at a dose of 1.8 g/kg of body weight/d. The collagen content in the granuloma was also significantly increased. In the case of the collagen profile, the pepsin-solubilized collagen content and its relative percentage to the total collagen were significantly higher than in the control. Histochemical examination showed that the granuloma tissues were well developed, and displayed many newly synthesized capillary vessels and a greater quantity of fibroblasts and monocytes in the 1.8 g leaf group. High density lipoprotein (HDL)-cholesterol and triglyceride content in the blood plasma were significantly higher than in the control. These results show that granuloma maturation was accelerated and the energy was supplied from fatty acid metabolism. The administration of Eucommia ulmoides OLIVER leaf may be effective at speeding up the wound healing process.

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Biological activities of a series of 2β-substituted analogues of 1α, 25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D₃-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene ergulation of the hydroxyalkyl and hydroxyalkoxy 2β-substituted analogues were almost comparable to those of 1α, 25(OH)₂D₃, while the alkyl and alkenyl analogues were much les active than 1α, 25(OH)₂D₃. This study investigated the biological evaluation of a series of 2β-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2β-position of 1α, 25(OH)₂D₃.

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Solasodine glycosides were analyzed for the selection of higher-producing natural resources in the fruits of Solanum spp. by western blotting and ELISA using anti-solamargine monoclonal antibody (MAb). Western blotting of Solanum khasianum showed a correlation with the analysis by ELISA. The results indicated that the fruits of S. khasianum contained the highest level of solasodine glycosides, compared to other species having 53.43±3.90 μg/mg dry wt. The high level of solasodine glycosides in S. khasianum suggests that it may be suitable for commercial production.

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We investigated the effect of Sansohnin-to ( ?? ?? ?? ??, SAT) on changes of duration in sodium pentobarbital (PB)-induced sleeping time caused by five types of stress.SAT reevrsed shortened PB sleep in repeated cold stress or 45 min-restraint stress tests and the prolonged PB sleep in 120 min-restraint stress. SAT did not reverse the shortened PB sleep in the specific stress state caused by an alternating rhythm in temperature stress or social isolation stress. In addition, SAT influenced both shortened PB sleep in 45 min-restraint stress and prolonged PB sleep in 120 min-restraint stress. SAT had no effect on PB sleep in unstressed control mice. These findings suggest that SAT has unusual activity, different from synthetic narcoleptics such as benzodiazepine. This is because SAT had no effect on PB sleep in unstressed mice, and it reverses stress-induced decrease and/or increase in PB sleep by improving stress-induced functional changes in the central nervous system, rather than by acting like a synthetic hypnotic on the γ-aminobutyric acid_A (GABA_A) receptor.

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Tranilast (TL) oily gels consisting of hydrogenated soybean phospholipid and fatty- acid ester were prepared, and the inhibitory effect of the gels on the growth of granulation tissue were evaluated in a carrageenin-induced rat granulation model. By the application of 0.1 and 0.2% TL oily gel, the weight of granulation tissue was significantly reduced to 64 and 55%, respectively, of control value. Furthermore, these gels reduced their respective hydroxyproline content to 64 and 51% of the control. On the other hand, the inhibitory effect of 10% TL ointments, which are clinically used for the treatment of keloids and hypertrophic scars as hospital preparations, was much lower than that of the oily gels. In addition, the application of 0.1 and 0.2% oily gel led to high concentration (0.1% gel, 168±18 μg/g; 0.2% gel, 221±16 μg/g) of TL in the dermis as compared with the 10% TL ointments.These results suggest that TL oily gels may be a useful topical formulation for the treatment of keloids and hypertrophic scars.

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Pulmonary absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) with various surfactants and protease inhibitors were examined in rats. The relative bioavailabilities of rhG-CSF with surfactants, such as polyoxyethylene 9-lauryl ether (Laureth-9) and sodium glycocholate (SGC), after intratracheal (i.t.) administration by intravenous (i.v.) and subcutaneous (s.c.) means were 37% (i.v.), 88% (s.c.), 84% (i.v.) and 197% (s.c.), respectively. These values were evaluated from the ratio of the area under the curve (AUC) of the plasma rhG-CSF concentration versus time for 8h. In the presence of various kinds of protease inhibitors, such as (p-amidinophenyl) methanesulfonyl fluoride·HCl (p-APMSF), aprotinin and bestatin, and increase in the plasma rhG-CSF concentration was observed, and the effect with p-APMSF was maximal. The relative bioavailabilities of rhG-CSF with p-APMSF after i.t. administration by i.v. and s.c. means were increased about 2-fold. To clarify the absorption mechanism of rhG-CSF, rhG-CSF was intratracheally administered with both Laureth-9 and p-APMSF. The AUC of rhG-CSF increased with both agents, and was approximately equal to that with SGC, which has both an enhancing effect on membrane permeation and an inhibitory effect on enzymatic degradation after i.t. administration. Consequently, it was considered that permeation and enzymatic degradation were rate-determining steps in the pulmonary absorption of rhG-CSF after i.t. administration.

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A novel method of assessing the extent of oral bioavailability of arginine-vasopressin (AVP) from pharmacological data was presented. After intravascular administration (i.v. bolus or short-term infusion) of AVP to rats, the relationship between blood concentrations and its effect on both mean arterial pressure (hemodynamic effect) and urinary sodium concentration (anti-diuretic effect) was described on the basis of an integrated pharmacokinetic-pharmacodynamic (PK-PD) model. A direct model was used for the hemodynamic response, while an indirect response model, rather than a hypothetical link model was used for the anti-diuretic response. A sigmoid E_max model was applied to describe the drug-receptor interaction. Pharmacological responses after intravascular administration of AVP were reasonably described by the PK-PD model. However, PD parameters estimated by the PK-PD analysis suggested that apparent receptor affinity rather than efficacy in i.v. bolus study was significantly higher than that in the short-term infusion study. This fact indicated that PK-PD relationship was influenced by the intravascular input rate of AVP. We than investigated the relationship between plasma concentration and amount of AVP bound to the V₂ receptors in the kidney. The result indicated that the amount of AVP bound to the receptors after i.v. bolus injection was always greater than that after short-infusion. Since the PK-PD relationship after oral administration was almost identical with that after short-term infusion, the PK-PD model obtained in the short-term infusion study was used to assess the extent of oral bioavailability (EBA_p.o.). The EBA_p.o. values, estimated from pharmacological effects (hemodynamic effect and anti-diuretic effect) after oral administration of 5μg/kg of AVP were 0.68% to 0.93% and were almost identical with the actual EBA_p.o. value (0.81%). From these results, we concluded that oral bioavailability of AVP was reasonably predicted by the PK-PD model, provided that appropriate pharmacological effects and appropriate intravascular dosing rate as a reference formulation are available. The method may be an alternative to methods based on plasma concentrations, when drug concentration cannot be measured and when appropriate pharmacological data are available.

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Lymphography, especially imaging of profundus lymph nodes, is a useful tool for diagnosis of cancer metastases in lymph nodes. However, positive enhancement agents for magnetic resonance lymphography (MRL) have not been available, since the positive imaging agents so far introduced are low-molecular-weight materials that are not trapped in lymph nodes. For the purpose of improved positive enhanced MRL, we employed liposomes as carriers of a positive enhancer, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). Magnetic resonance (MR) imaging was performed after subcutaneous injection of Gd-liposomes into the hind feet of rabbits which had reactive enlarged retroperitoneal lymph nodes. As results, not only popliteal but also profundus retroperitoneal lymph nodes were positively enhanced by Gd-liposomes, especially after 20 min massage of the injected sites. Gd-Liposomes containing dipalmitoylphosphatidylglycerol were more effective than Gd-liposomes containing palmityl-D-glucuronide, a type of long-circulating liposomes, suggesting that liposomal accumulation in lymph node is, least partly, mediated by the trapping of liposomes by macrophages. These data show that liposomes modified with Gd-DTPA are effective for positive enhancement of both regional and profundus lymph nodes in MR lymphography.

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Hydroxyproline (Hyp) content in biologial fluids is used as a parameter of collagen catabolism, especially bone resorption. In this paper, we examined the relationship between agin and serum free 4-Hyp and proline (Pro) content using a newly introduced analytical technique which is chemiluminescence determination with electrogenerated tris (2, 2'-bipyridine) ruthenium(III). Serum was collected from 50 bedridden aged people and 131 normal subjects living in Yamato-son, Amamioshima, Kagoshima. In the free 4-Hyp content of normal serum, the change in females is more gentle than that males with aging. The free 4-Hyp content of bedridden aged serum is significantly (P<0.05) elevated compared to normal aged serum. Bedridden aged females and males also show levels significantly (p<0.05) elevated in comparison to normal. These changes are due to bone resorption based on bedridden patients' atrophy. In bedridden aged people, free 4-Hyp of those with a fracture was significantly (p<0.05) elevated than in people without a fracture. No relationship between low bone density and high free 4-Hyp in serum was observed. It seems that bone resorption or bedridden atrophy may be enhanced in bedridden aged people by a fracture. In free Pro content in serum, no significant change was observed. These results show that the free 4-Hyp content in serum is useful in the clinical or biological inspection of bone resorption, especially in aged people.

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The peak of glycyrrhizin (GL) β-D-glucuronidase activity for Ruminococcus sp. PO1-3 and Eubacterium sp. GLH changed to 24 h from 12 h of culture and to 12 h from 48 h, respectively, at almost the same level by the addition of 1.0 mM GL. This enzyme activity was about 20-fold higher in Eubacterium sp. GLH than in Ruminococcus sp. PO1-3. GL β-D-glucuronidase activity of Ruminococcus sp. PO1-3 with Eubacterium sp. GLH and the intestinal flora showed a maximal peak at 12 h of culture in the presence and absence of 1.0 mM GL. This enzyme activity was about 2.5-fold higher in mixed bacteria than in intestinal flora. 3β-Hydrosteroid dehydrogenase activity of Ruminococcus sp. PO1-3 and Ruminococcus sp. PO1-3 with Eubacterium sp. GLH was suppressed greater in the presence of GL than without GL. Also, Ruminococcus sp. PO1-3, Eubacterium sp. GLH, and a mixture of both and intestinal flora, metabolized 1.0 mM GL to glycyrrhetic acid (GA) in yields of about 10, 70, 40 and 100%, respectively, with 24 h culture. From the level of GL β-D-glucuronidase activity, it is considered that the metabolism of GL by intestinal flora is due to both enzymatic and non-enzymatic reactions. Moreover, GA at a concentration of 1.0 mM suppressed growth of Ruminococcus sp. PO1-3, Eubacterium sp. GLH, and the mixture of both and intestinal flora, which metabolized 1.0 mM GA to a negligible amount of 3-oxo-glycyrrhetic acid, indicating the accumulation of unchanged GA. GL β-D-glucuronidase activity of intestinal flora was enhanced by GA, which stimulated bactria possessing particular this characteristic.

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We studied the biosynthetic route of thiamin in Saccharomyces cerevisiae to see whether the route differed under aerobic and anaerobic conditions.Histidine and pyridoxine are the precursors of the pyrimidine moiety of thiamin under aerobic conditions. Formate is incorporated into the pyrimidine via histidine. The incorporation of [¹³C]formate and [5'-²H₂]pyridoxine into the pyrimidine was wxaminied under anaerobic donritions. The labels from [¹³C]formate and [5'-²H₂]pyridoxine were not incorporated into the pyrimidine under anaerobic conditions, indicating that the biosynthetic pathway of the pyrimidine differed from that under aerobic conditions.On the other hand, [¹⁵N]glycine was incorporated into the thiazole under both anaerobic and aerobic conditions. The biosynthetic pathway of the thiazole was therefore unaltered by the O₂ concentration.

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This study demonstrates that astemizole, a non-sedating anti-histaminergic drug low toxicity in vivo, greatly potentiates the growth-inhibitory activity of doxorubicin in doxorubicin-resistant human leukemia cells (K562/DXR). Astemizole synergistically potentiated the cytotoxicity of doxorubicin for K562/DXR cells at a concentration of 0.1-3 μM in a dose- dependent manner, whereas they showed hardly any synthergistic effect in the parental cell line (K562) at the same concentration. Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of astemizole on P-glycoprotein activity in cytoflurographic efflux experiments with doxorubicin. Our results indicate that astemizole inhibits the P-glycoprotein pump-efflux activity in a dose-related manner. moreover, it also inhibits the photolabeling of P-glycoprotein by [³H]azidopine in a dose-dependent manner. These findings provide a biological basis for the potential therapeutic application of astemizole as an anticancer drug either alone or in combination with doxorubicin to multidrug-resistant leukemic cells.

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We examined the role of endotoxin in the mechanism of recombinant human tumor necrosis factor (rhTNF)-hypersensitivity caused by D-galactosamine (GalN). We used polymyxin B, an antibiotic with anti-endotoxin activity, to determine the participation of endogenous endotoxin. The glycogen and blood glucose level of rhTNF (1×10⁴ units/mouse, i.v.)-injected mice was lower at 7 h post-intoxication than that in the control. Administration of rhTNF to GalN (700 mg/kg, i.p.)-treated mice resulted in lower levels of glycogen and blood glucose than those in animals treated with rhTNF alone. In mice pretreated with polymxin B (20 mg/kg, i.p.), the level at 7 h after rhTNF/GalN-injection was markedly increased compared to that in mice treated with rhTNF/GalN alone. The injection of a low endotoxin dose (0.1 mg/kg, i.p.) markedly decreased the rectal temperature in mice treated with rhTNF (5×10³ units/mouse, i.v.) and GalN, and none of these animals survived after treatment for 18 h. These findings suggest that endogenously produced endotoxin may contribute to the extent of rhTNF-hypersensitivity caused by GalN.

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We recently investigated the relationship between the structures of various indenestrols and their cytotoxicity, and reported that indenestrol A (IA), a metabolite of the synthetic nonsteroidal estrogen diethylstilbestrol, and indenestrol B (IB), an analog of IA, disrupt the microtubule architecture of Chinese hamster V79 cells in vitro. We then synthesized 16 optically active indenestrol derivatives by substituting monoethyl, monobenzyl and diethyl ether groups at the 6- and/or 4'-hydroxyl positions, and examined their cytotoxic activities in Chinese hamster V79 cells. The results indicated that the monoethyl ethers had cytotoxic activities similar to monomethyl ehters. However, the (+)- and (-)-monobenzyl ethers were less cytotoxic than the corresponding monomethyl and monoethyl derivatives.

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As a part of our search for bioactive substances from the leaves of Perilla frutescens BRITTON var. acuta KUDO (Perillae Herba, Labiatae), the aqueous extract was orally administered to rats and humans, and metabolites in the urine, plasma, and/or bile were analyzed by a high-performance liquid chromatograph (HPLC) equipped with a photodiode array detector. When the extract was administerd to rats, 10 metabolites, trans-caffeic acid-4-O-sulfate (1), trans-p-coumaric acid-4-O-sulfate (2), trans-ferulic acid-4-O-sulfate (3), trans-m-coumaric acid-3-O-sulfate (4), trans-caffeic acid (5), m-hydroxyphenylpropionic acid (6), trans-p-coumaric acid (7), trans-m-coumaric acid (8), luteolin (9), and apigenin (10) were detected in the urine, whereas four metabolites, scutellarein-6, 7-di-O-β-glucuronide (11), apigenin-4'-O-sulfate-7-O-β-glucuronide (12), apigenin-7-O-β-glucuronide (13), and diosmetin-7-O-β-glucuronide (14) were found in the bile. Compounds 1-8 and 11-14 were also found in the plasma. When the extract was given to humans, however, two metabolites, 1-O-(2, 4, 5-trimethoxycinnamoyl)-β-glucuronic acid (15) and apigenin-4'-O-β-glucuronide (16), were found in the urine and plasma. Thus, a species difference in the metabolism of the extract constituents was observed between rats and humans. Structures 1-16 were identified based on their chemical and spectral data.

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Glucuronides of RT-3003 and its metabolite (9-OH-RT-3003), which was hydroxylated at the 9 position on the benzene ring, were separated by HPLC and identified by liquid chromatography (LC)/MS/MS and NMR. The conjugation sites of these glucuronides were determined by nuclear Overhauser effects (NOE) irradiation; RT-3003 was conjugated at an alcoholic hydroxyl group of the hydroxymethyl moiety, and 9-OH-RT-3003 at a phenolic hydroxyl group on a benzene ring and at an alcoholic hydroxyl group of a hydroxymethyl moiety. On a reversed-phase HPLC of 9-OH-RT-3003, alcoholic glucuronide was eluted later than phenolic glucuronide, indicating the high hydrophobicity of alcoholic glucuronide. Clearance for the glucuronidation (Cl_G) of RT-3003 was lower than the summation of Cl_G for two types of glucuronidation of 9-OH-RT-3003. Cl_G of 9-OH-RT-3003 was high in phenolic glucuronide. The activity of UDP-glucuronyltransferase (UDPGT) for RT-3003 was 9.63 times that for 9-OH-RT-3003, and the activity ratio of the two types of glucuronidation of 9-OH-RT-3003 was similar to the ratio of the corresponding Cl_G. The difference between Cl_G and UDPGT activity is discussed in association with clearance for the hydroxylation and interaction of substrates with UDPGT.

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