The objective of this study was to investigate the applicability of new oxygen meters with multi-channels and disposable oxygen electrode sensors (DOX-96) on the antimicrobial susceptibility testing of clinical bacterial isolates. The oxygen amount in the wells of 96-well plates was converted into current through electrodes. Bacterial inoculation decreased the oxygen amount in the wells because viable bacteria consume the oxygen. On the other hand, a failure of bacteria to consume oxygen was observed in the presence of potent antimicrobial agents, representing a serious arrest of bacterial metabolism usually leading to stasis or death. Based on these results, the minimum inhibitory concentration was determined by DOX-96 (MICdox). The MICdox showed good agreement with MIC measured by the standard broth microdilution method (98.2%). DOX-96 was also useful for turbid samples such as Mueller-Hinton broth containing 0.1% lipid emulsion. The MICdox in turbid samples showed good agreement with those in clear samples (100.0%). These results indicate that the newly developed DOX-96 is very useful in antimicrobial susceptibility testing even in turbid clinical samples such as colloidal products and turbid biological components.

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Matrix metalloproteinases (MMPs), especially membrane-type 1 matrix metalloproteinase (MT1-MMP), which generates an active form of MMP-2 from proMMP-2, are deeply involved in angiogenesis as well as in tumor cell migration and metastasis. To obtain a specific inhibitor for MT1-MMP, we screened a number of natural and synthetic compounds using recombinant human MMP-2, MMP-7, and soluble MT1-MMP in a fluorogenic peptide cleavage assay. (−)-Epigallocatechin 3-O-gallate (EGCG) followed by (−)-epigallocatechin 3,5-di-O-gallate and epitheaflagallin 3-O-gallate, was found to have potent and distinct inhibitory activity against MT1-MMP. Therefore, we investigated the effect of EGCG on the suppression of MMP-2 activation as determined by gelatin zymography, and observed that the active form of MMP-2 in the conditioned medium of human umbilical vein endothelial cells was decreased in the presence of EGCG. The results suggest the possibility that tea polyphenols suppress tumor growth through the suppression of angiogenesis.

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The effect of supplementation of n-3 polyunsaturated fatty acids (PUFA) on oxidative stress-induced DNA damage of rat hepatocytes was examined. Male Wistar rats were fed a diet containing safflower oil (control n-6 PUFA diet) or fish oil (n-3 PUFA diet) in 50 g/kg of dried diet and an equal amount of vitamin E in 59 mg/kg of dried diet for 6 weeks. The liver of rats fed safflower oil was rich in n-6 PUFA, whereas that of rats fed fish oil was rich in n-3 PUFA. Isolated hepatocytes were treated in vitro with ADP/Fe (II) ion or hydrogen peroxide at 37 °C for 30 min to induce oxidative stress. The degree of lipid peroxidation was assessed by the levels of phospholipid hydroperoxides and thiobarbituric acid-reactive substances. The degree of oxidative DNA damage was assessed based on comet-type characterization in alkaline single-cell gel electrophoresis and 8-hydroxy-deoxyguanosine levels. In both ADP/Fe(II) ion and hydrogen peroxide oxidation, the degree of lipid peroxidation of hepatocytes increased in both diet groups, and the level of increase in the fish oil diet group was slightly higher than that in the safflower oil diet group. In ADP/Fe(II) ion oxidation, the degree of DNA damage increased in both diet groups, but there were no significant differences in the level of increase. In contrast, in hydrogen peroxide oxidation, the degree of DNA damage increased in both diet, and the increase in the fish oil diet group was significantly lower than that in the safflower oil diet group. It is unlikely that an n-3 PUFA-rich diet enhances oxidative stress-induced hepatocyte DNA damage as compared with the control n-6 PUFA-rich diet.

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We examined whether fluvastatin (FV), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has antioxidant activity against oxidative DNA damage to hamster pancreas induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP). Female Syrian golden hamsters were treated with FV by gastric intubation 30 min before BOP administration. Control animals were intubated with saline. Animals were injected subcutaneously with BOP (20 mg/kg body weight) or saline, and sacrificed 1 and 4 h later. The contents of 8-oxo-2′-deoxyguanosine (8-oxodG) in the nuclear DNA and thiobarbituric acid-reacting substances (TBARS) were measured in the pancreas and liver of hamsters. Treatment with more than 0.22 mg/kg body weight FV significantly inhibited the increase in 8-oxodG content induced by BOP treatment. The TBARS contents in pancreas changed similarly by intubation of FV. In the liver, the contents of 8-oxodG and TBARS were not affected by a single administration of BOP. The protective effect of FV was stronger than those of pravastatin and other antioxidants such as Trolox, ascorbic acid and green tea catechin. These results suggest that FV inhibits oxidative damage to DNA and lipids caused by reactive oxygen species formed through the metabolism of chemical carcinogens.

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Novel endotoxin-tolerance was observed to the cytotoxycity induced by lipopolysaccharide (LPS) and cycloheximide (CHX) in an LPS-treated macrophage-like cell line, J774.1; preincubation of macrophages with low doses of LPS alone for 90 min almost completely prevented the apoptotic death in the second incubation with LPS and CHX. The first challenge of LPS affected neither the subsequent LPS binding nor the expression of CD14. Instead, phosphorylation of mitogen-activated proteinkinase (MAP kinases) involving p38, extracellular signal-regulated kinase 1/2 (Erk1/Erk2) and c-jun N-terminal kinase (JNK) in the second incubation with LPS and CHX were suppressed, suggesting that this endotoxin-tolerance was caused by down-regulation of LPS-signaling pathway leading to MAP kinase activation. On the other hand, LPS-induced cytotoxicity seemed to depend on the sustained phosphorylation of p38 MAP kinase; the addition of SB202190, an inhibitor of p38 MAP kinase activity, in the first incubation with LPS caused induction of the cytotoxicity in the second incubation with LPS and CHX or CHX alone, under which conditions increased phosphorylation of p38 MAP kinase without that of Erk1/Erk2 or JNK was observed. These results suggest that down-regulation of the p38 MAP kinase cascade in the first incubation with LPS is linked to induction of endotoxin-tolerance to the cytotoxicity with higher doses of LPS and CHX.

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Mast-cell-deficient WBB6F1-W/W^v mice (W/W^v) and congenic wild-type (+/+) mice were sensitized by oral administration of 0.1 or 1.0 mg ovalbumin (OVA) in the form of gavage every day for 9 weeks, and active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of OVA. Production of OVA-specific IgG1 in response to oral sensitization of the W/W^v mice was very high, and the production of IL-4, IL-5 and IL-10 by splenocytes re-stimulated with OVA in vitro was increased. These findings suggest that Th2-dominant helper T-cell activation had occurred. By contrast, production of OVA-specific IgG1 was low in +/+ mice, and no significant increase in production of Th2-type cytokines by the splenocytes of +/+ mice was observed. Population analysis in Peyer's patches by flow cytometry revealed that the proportion of the CD11c+ cell in the W/W^v mice was slightly increased after antigen stimulation. Analysis of the cell surface markers of intraepithelial lymphocytes (IELs) by flow cytometry showed that the proportion of TCRγδ-T cells was extremely lower in the W/W^v mice, especially in the antigen sensitized group. The proportion of TCRγδ-T cells in the splenocytes of W/W^v mice was also lower than in +/+ mice. Taken together, the above findings indicate that W/W^v mice seems to be a good model not only for studying the induction mechanism of food allergy but for examining the role of TCRγδ-T cells in food-induced hypersensitivity.

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The aryl hydrocarbon receptor (AhR) mediates a spectrum of toxicological and biological effects of dioxins. Studies on tissue distribution of the AhR in developing and adult animals demonstrated that the AhR is expressed in a tissue-specific and developmentally specific manner. Also, the expression level of the AhR in culture cells varies more than 50-fold among cell lines. Although the mode of AhR action has been studied extensively, the events that control the expression of the AhR gene itself are still poorly understood. We previously showed that the AhR protein is depleted during adipose differentiation, resulting in the loss of functional response to xenobiotics. In this study, to understand the mechanism by which the AhR is depleted during adipogenesis, we analyzed the AhR promoter activity during adipose differentiation in 3T3-L1 cells. Nuclear run-on assay revealed that the downregulation of the AhR during adipogenesis is primarily at the transcriptional level. To identify the sequence of the AhR promoter region responsible for differentiation-dependent suppression of AhR transcription, a series of deletion constructs linked to the CAT reporter were transfected into 3T3-L1 cells. A comparison of CAT activity between preadipocytes and adipocytes revealed that the sequence −378/−359 is core contributor to differentiation-dependent downregulation of AhR promoter activity. EMSA and UV crosslinking studies showed the presence of the factor bound to the sequence −378/−359. The binding activity was apparently higher in preadipocytes than in adipocytes. Consequently, the downregulation of the trans-acting factor may result in the suppression of AhR gene transcription during adipose differentiation.

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The liver contrast effects of Sonazoid in two ultrasonographic imaging modes, gray-scale conventional and harmonic, were examined as a time-related study in normal rabbits, and evaluated quantitatively and visually with tumor-model rabbits to estimate the diagnostic potential. Peak enhancement of vessels and parenchyma was observed 1 min after injection in both modes, although signal enhancement in the parenchyma lasted for 120 min compared with rapid decay (5—10 min) in vessels. When Sonazoid was intravenously injected into metastatic carcinoma-model (VX-2) rabbits, all hepatic tumors showed ring enhancement in the early phase followed by clear contrast defects in the delayed phase, because signal enhancement remained only in normal parenchyma. Visual analysis scores for the diagnosis of tumors were improved by Sonazoid injection, and the videodensitometric differences between tumor and normal tissues were significantly greater after injection. Although the harmonic mode tended to show better contrast effects, the conventional mode provided significant contrast enhancement in this hepatic tumor-model. Sonazoid might be useful for the detection of undifferentiated tumors in the liver by making it possible to visualize neovascularity in the early phase and clear contrast defects in the delayed phase, not only in the harmonic but also in the conventional mode.

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The anti-inflammatory effect of the leaves of Bryonia laciniosa was evaluated using carrageenan, dextran, histamine, serotonin induced rat paw oedema and cotton pellet induced granuloma (chronic) models in rats. In mice, carrageenan peritonitis test was performed for the extract by oral administration. The chloroform extract of Bryonia laciniosa (CEBL) exhibited significant anti-inflammatory effect at the dose 50, 100 and 200 mg/kg. Maximum inhibition (52.4%) was noted at the dose of 200 mg/kg after 3 h of drug treatment in carrageenan induced paw oedema, whereas the indomethacin (standard drug) produced 62.1% of inhibition. The extract exhibited significant anti-inflammatory activity in dextran induced paw oedema in a dose dependent manner. The extract also exhibited significant inhibition on the hind paw oedema in rats caused by histamine and serotonin respectively. In the chronic model (cotton pellet induced granuloma) the CEBL (200 mg/kg) and standard drug showed decreased formation of granuloma tissue by 50.1 and 57.3% (p<0.001) respectively. The extract also inhibited peritoneal leukocyte migration in mice. Thus, the present study revealed that the chloroform extract of Bryonia laciniosa exhibited significant anti-inflammatory activity in the tested models.

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Butein (3,4,2′,4′-tetrahydroxychalcone), a plant polyphenol, has been known to elucidate endothelium-dependent vasodilation. In the present study, the hypotensive effect of butein and its possible mechanism, especially an angiotensin converting enzyme (ACE) inhibitory effect, were investigated. Intravenous injection of butein lowered the arterial blood pressure of anesthetized rats in a dose-dependent manner. The plasma ACE activities were significantly inhibited by the addition of butein in a dose-dependent manner, the IC₅₀ value of which was 198 μg/ml (730 μM). Moreover, angiotensin I-induced contraction was markedly attenuated by prior exposure of endothelium-intact aortic rings to butein, but angiotensin II-induced contraction was not altered. These results suggest that butein has a hypotensive effect, at least in part, via the inhibition of angiotensin converting enzyme.

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For the purpose of the development of a skin-whitening agent, Sophora flavescens was evaluated for tyrosinase inhibitory activity and its active principles were identified following activity-guided isolation. The ethanol extract and dichloromethane fraction from S. flavescens showed significant inhibition of mushroom tyrosinase. From the dichloromethane fraction, three known prenylated flavonoids, sophoraflavanone G, kuraridin, and kurarinone, were isolated. Compared with kojic acid (IC₅₀=20.5 μM), these compounds possessed more potent tyrosinase inhibitory activity. The IC₅₀ values were 6.6, 0.6, and 6.2 μM for sophoraflavanone G, kuraridin, and kurarinone, respectively.

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After bioassay-guided fractionation of the extract from Sandoricum koetjape bark, which exhibited significant toxicity to killifish (Oryzias latipes), two ichthyotoxic triterpenoids were isolated and characterized as koetjapic acid and 3-oxo-olean-12-en-29-oic acid. These constituents, along with non-toxic katonic acid, had a remarkable inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA), which is a preliminary in vitro screening method for possible anti-tumor-promoting agents. Of the triterpenoids active in vitro, koetjapic acid appears to be a promising cancer chemopreventive agent, since it significantly delayed tumor promotion in two-stage mouse skin carcinogenesis induced by 7,12-dimethylbenz(a)anthracene and promoted by TPA.

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In the present study, a series of 6-bromo-2,3-disubstitued-4(3H)-quinazolinones was synthesized by condensation of 6-bromo-2-substituted-benzoxazin-4-one with trimethoprim, pyrimethamine and lamotrigine. The chemical structures of the synthesized compounds were confirmed by means of IR, ¹H-NMR and mass spectral and elemental analysis. The antiviral activity and cytotoxicity of the compounds were tested in E₆SM (Herpes simplex-1 KOS, Herpes simplex-1 TK-KOS ACV, Herpes simplex-2 G, Vaccinia virus, Vesicular stomatitis virus, Parainfluenza-3 virus, Reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta Toro virus) and HeLa cell culture (Vesicular stomatitis virus, Coxsackie virus B4 and Respiratory syncyticla virus). Investigation of anti-HIV activity was done against replication of HIV-1 (HTLV-III B LAI) in MT-4 cells. 6-Bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the most potent antiviral activity with a MIC of 1.92 μg/ml against vaccinia virus in E₆SM cell culture. The other compounds did not exhibit antiviral activity nor afford significant cytoprotection to the E₆SM and HeLa cell culture when challenged with the viruses. The study implies that 4 may possess activity against Pox viruses including variola. In the anti-HIV study, 6-bromo-2-methyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (3) and 6-bromo-2-phenyl-3-[(4-amino-5-(4-chlorophenyl)-6-ethylpyrimidin-2-yl]-4(3H)-quinazolinone (4) exhibited the least cytotoxic concentration (0.424, 0.461 μg/ml) which is an index of the infective viability of mock infected MT-4 cells with HIV-1. None of the compounds exhibited significant anti-HIV activity.

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To characterize the antinociceptive profiles of Angelica gigas NAKAI (ANG; Korean angelica), methanol extract from the dried roots of ANG was made and mice were administered orally at the various doses (from 0.25 to 3 g/kg). ANG produced the increased latencies of the tail-flick and hot-plate paw-licking responses in a dose-dependent manner. In acetic acid-induced writhing test, ANG dose-dependently decreased writhing numbers. Moreover, the cumulative response time of nociceptive behaviors induced by intraplantar formalin injection was reduced during both the 1st and the 2nd phases in a dose-dependent manner in ANG-treated mice. Furthermore, oral administration of ANG did not cause licking, scratching and biting responses induced by TNF-α (100 pg), IFN-γ (100 pg) or IL-1β (100 pg) injected intrathecally (i.t.), especially at higher dose (3 g/kg). Additionally, in ANG treated mice, the cumulative nociceptive response time for i.t. administration of substance P or capsaicin was dose-dependently diminished. Finally, nociceptive responses elicited by i.t. injection of glutamate (20 μg), N-methyl-D-aspartic acid (60 ng), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (13 ng) or kainic acid (12 ng) were decreased by oral administration of ANG. Our results suggest that ANG produces antinociception via acting on the central nervous system and shows antinociceptive profiles in various pain models, especially inflammatory pain.

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A reliable enzyme immunoassay (EIA) method was developed for quantitative determination of aconitine with high sensitivity and specificity. The bovine serum albumin (BSA)- and β-galactosidase (β-Gal) conjugates as immunogens and enzyme-labeled antigens were prepared by coupling of their proteins with succinic acid (short chain length; n=2, where n represents the number of methylene units) and hexadecanedioic acid (long chain length; n=14) hemiesters of benzoylaconine through the respective N-hydroxysuccinimide esters as intermediates. Two types of the BSA-conjugates with short and long chains were repeatedly injected into rabbits to obtain anti-aconitine antisera (As1 and As2, respectively). All combinations of β-Gal-labeled antigens LAg1 (n=2) and LAg2 (n=14) with antisera As1 (n=2) and As2 (n=14) showed high sensitivity to aconitine in a range of 0.1—1.0 ng. Although the combination of LAg2 (n=14) with antiserum As1 (n=2) showed high specificity to aconitine, the combination of LAg2 (n=14) and As2 (n=14) was highly specific to both aconitine and mesaconitine. When aconitine was intravenously administered to rats, the aconitine concentration in their plasma remarkably decreased within the first 60 min, and then gradually declined, suggesting a two-compartment pharmacokinetic model in (V_c 0.41±0.09 l/kg, V_dss 1.7±0.4 l/kg, CL_tot 10±2 ml/min · kg, AUC_0—4800 2055±294.3 ng · min/ml). Following oral administration of aconitine to rats at two doses of 0.1 and 1.0 mg/kg b.w., the maximum plasma concentrations (C_max) were 0.73±0.08 and 3.3±0.6 ng/ml at times of 45±9 and 150±52 min, respectively, and the AUC_0—1440 values were 130±4 and 1600±270 ng · min/ml. The bioavailability (F) of aconitine was determined to be 0.013, where only 1.3% of the aconitine administered orally was absorbed into the body fluid.

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Dimethyl sulfoxide (DMSO) has anti-inflammatory and analgesic properties and is the only intravesical agent approved by the FDA for the treatment of interstitial cystitis. While it is known that DMSO has numerous biological effects on cell differentiation and alteration of cell-surface carbohydrate structures, the anti-inflammatory mechanism of DMSO has been not clear yet. Therefore, further investigation of DMSO in terms of inflammation therapy is needed. This study assessed the in vitro anti-angiogenic effects of DMSO on human aorta endothelial cells to clarify one of the mechanisms of its anti-inflammatory activity. DMSO did not affect expression of E-selectin on endothelial cells in the presence of TNF-alpha. Furthermore, DMSO effectively inhibited capillary tube formation; this mechanism would be due to suppression of matrix metalloproteinase-2 (MMP-2) production. These results provide useful knowledge about the anti-inflammatory effects of DMSO and the regulatory mechanism of MMP-2.

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The rhizome of Dryopteris crassirhizoma NAKAI exhibited significant antioxidant activity, as assessed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity in vitro. Two phloroglucinol derivatives, flavaspidic acids PB (1) and AB (2), were isolated from the rhizome of D. crassirhizoma by a bioassay-guided fractionation. ¹H-, ¹³C-NMR, and UV analysis were used to determine the structures. Furthermore, the two compounds were tested for their antioxidant activities, such as their DPPH radical scavenging, superoxide radical scavenging, and lipid peroxidation (LPO) inhibitory activities. Compounds 1 and 2 exhibited potent antioxidant activity against the LPO inhibitory test with IC₅₀ values of 12.9 and 13.1 μM, respectively, compared with α-tocopherol (IC₅₀; 15.6 μM) and butylated hydroxy anisole (BHA, IC₅₀; 10.8 μM), while the two compounds had a moderated effect on the DPPH radical scavenging activity (IC₅₀; 71.7, 76.3 μM) as well as superoxide radical scavenging activity (IC₅₀; 58.6, 64.4 μM). The potent activity of the flavaspidic acids (1, 2) on inhibiting LPO might be due to possible stabilization as a result of chelating with iron.

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The effects of soyasapogenol B, sophoradiol, their glucuronides, and glycyrrhizin on the hepatotoxicity of tert-butyl hydroperoxide (t-BuOOH) in a human-liver-derived cell line (HepG2 cells) were investigated. Glycyrrhizin showed significant dose-dependent protective effects against the cytotoxicity of t-BuOOH. Among soyasapogenol B and its glucuronides, the monoglucuronide showed the most potent hepatoprotective activity, followed by soyasapogenol B itself. Soyasaponin III was weakly protective, while soyasaponin I increased the toxicity of t-BuOOH. Among sophoradiol and its glucuronides, sophoradiol itself showed the most potent hepatoprotective activity, which was equal to glycyrrhizin, while the monoglucuronide and kaikasaponin III showed an increase in cytotoxicity. These results were considerably different from those reported previously on the protective effects of these compounds using primary cultures of immunologically injured rat liver cells. Consequently, the hepatoprotective action of the triterpene derivatives investigated would be different in HepG2 cells and in rat primary hepatocyte cultures.

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Adenine was isolated as a platelet aggregating inhibitor from the leaves of Cassia alata by HPLC using a triacontylsilyl silica (C₃₀) column. The inhibitory effects of adenine and adenosine (positive control) on the platelet aggregation induced by collagen or adenosine 5′-diphosphate (ADP) as an aggregating agent was evaluated with a platelet aggregometer using a laser-scattering method. As a result, the inhibitory effect of adenine was observed in the platelet aggregation induced by collagen (1.0 μg/ml as the final concentration), but little inhibitory effect was noted in the aggregation induced by ADP (5.0 μM as the final concentration), whereas adenosine exhibited potent inhibitory effects on platelet aggregation induced both by collagen and ADP under the same experimental conditions.

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We developed a simple method for preparing a highly concentrated solution of glycyrrhizin monoammonium salt (GZ) at low viscosity with no surfactants nor organic solvents and investigated the absorption profile after rectal administration to rats. GZ (200 mg/ml) was dissolved in phosphate buffered solution, pH 7.0; over 350 mM concentration was maintained for the aqueous solution without gel-formation. When glycerin was used as a non-aqueous formulation, GZ did not form gel. Apparent permeability coefficients of GZ obtained from 350 mM phosphate buffered solution (pH 7.0) and glycerin solution through rat rectal mucosa estimated by in vitro parallel diffusion chamber technique were 0.686×10⁻⁶ and 0.379×10⁻⁶ cm/s, respectively. On the other hand, the area under plasma concentration–time curves of GZ in 400 mM phosphate buffer (pH 7.0) and glycerin formulations after rectal administration to the rat were significantly higher than that in polyethylene glycol 400/propylene glycol (55 : 5) formulation. Maximum plasma concentrations of these formulations were dependent on the apparent permeability coefficients of GZ. Increased absorption observed by phosphate buffered formulation accompanied no pronounced histological damage in mucosa. These results demonstrate that addition of a highly concentrated phosphate salts is effective not only for lowering the viscosity of a highly concentration of GZ solution, but also for improving the mucosal GZ absorption.

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We investigated the transfection efficiency mediated by asialoganglioside-containing cationic liposomes. Previously we reported that monosialoganglioside GM₁ (GM_1a) enhanced transfection efficiency. In this study, we investigated the effects of sialic acid in gangliosides on transfection efficiency. Two mammalian culture cell lines HeLa and HepG2 were transfected with luciferase plasmids (pGL3) using cationic liposomes which contain monosialoganglioside GM₁ (GM_1a) or its asialic counterpart, asialoganglioside GM₁ (GA₁). Both GM_1a and GA₁ enhanced the efficiency of transfection mediated by cationic liposomes, and GA₁ exhibited higher efficiency than GM_1a in both cell lines. Transfection efficiency of ganglioside-containing liposomes was also assessed by the effects of antisense oligonucleotides (AS-ODN) for bcl-2 gene, which suppresses apoptotic cell death. Western blotting analysis revealed that the expression of Bcl-2 was decreased by AS-ODN, and the reduction of protein expression in cells treated with GA₁-containing liposomes was more remarkable than that with GM_1a-containing liposomes. Furthermore, the induction rate of apoptosis was higher in cells treated with AS-ODN with GA₁-containing liposomes. Together with the results obtained by luciferase assay mentioned above, the removal of sialic acid from ganglioside causes the enhancement of efficiency of transfection mediated by cationic liposomes.

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The age and species dependent characteristics of cutaneous esterase activity were examined in cultured keratinocytes of neonatal and adult humans and of rats at the age of 1, 3, 10, and 50 d. The existence of esterases was characterized using fluorescein-5-isothiocyanate diacetate under a confocal laser scanning microscope. In vitro hydrolysis of ethyl nicotinate (EN), an esterified prodrug of nicotinic acid, was investigated in homogenate of cultured keratinocytes, and the Michaelis–Menten parameters (V_max and K_m) of EN were evaluated. Together with development and growth of rats and humans, V_max and V_max/K_m increased drastically, suggesting that esterases in keratinocytes develop markedly during the growth process. The affinity parameter, K_m, was almost the same among the ages in each species. These findings in cultured keratinocytes corresponded with our previous report using dissected skin specimens. Species differences in V_max, V_max/K_m and K_m were also observed, and these parameters of EN hydrolysis in rats were significantly higher than that in humans. In conclusion, cultured keratinocytes can be an advantageous method with which to estimate cutaneous activation of ester prodrugs in humans and during the growth process.

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The effects of Ginkgo biloba leaf extract (GBE), one of the most widely used herbal dietary supplements in Japan, on the pharmacokinetics of diltiazem (DTZ), a typical probe of cytochrome P450 (CYP) 3A, were examined in rats. The simultaneous addition of GBE to small intestine and liver microsomes inhibited the formation of N-demethyl DTZ (MA), an active metabolite of DTZ produced by CYP3A, in a concentration-dependent manner, with an IC₅₀ of about 50 and 182 μg/ml, respectively. This inhibition appeared to be caused, at least in part, by a mechanism-based inhibition. Both the rate of formation of MA and total amount of CYP in intestinal or hepatic microsomes after a single oral pretreatment with GBE (20 mg/kg) decreased transiently. The pretreatment significantly decreased the terminal elimination rate constant and increased the mean residence time, after intravenous administration of DTZ (3 mg/kg). Furthermore, it significantly increased the area under the concentration–time curve and absolute bioavailability after oral administration of DTZ (30 mg/kg). These results indicated that the concomitant use of GBE in rats increased the bioavailability of DTZ by inhibiting both intestinal and hepatic metabolism, at least in part, via a mechanism-based inhibition for CYP3A.

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Ointments of the skin depigmentation agent hydroquinone (HQ) have been prepared by extemporaneous nonsterile compounding in our hospital. The HQ ointments were highly effective in the treatment of various types of skin pigmentations; however, various problems have emerged including chromatic aberration of the ointments, a relatively large variability of efficacy, and mild topical side effects including irritation. In this paper, the cytotoxicity of HQ was assessed in vitro using rat skin fibroblasts as the concentration with 50% survival after 24 h exposure to be 16.5 μM. The intradermal concentrations at 2 h after application of the HQ ointments was also estimated to be 358 mM and 51.7 mM in stratum corneum and viable tissue (viable epidermis+dermis), respectively, by an in vitro rat skin permeation study with rat full-thickness abdominal skin and Franz-type diffusion cells. It was demonstrated that the intradermal concentration of HQ was much higher than that eliciting cytotoxicity, suggesting that the topical side effects after application of HQ ointment were due to the cytotoxicity of HQ.

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The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^·+) decolorization assay was applied to evaluate the stoichiometric radical scavenging activity of ascorbic acid (AA) and two AA derivatives, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) and 2-O-α-D-glucopyranosyl-6-O-octanoyl-L-ascorbic acid (6-Octa-AA-2G). AA rapidly reacted with ABTS^·+, and the reaction was completed within 10 min. In contrast, AA-2G and 6-Octa-AA-2G continuously reacted with ABTS^·+, and the reaction was not completed after 2 h. The radical scavenging activity of AA-2G and 6-Octa-AA-2G in aqueous solutions at pH 4.0 and above was higher than that at pH 3.0, whereas AA showed no difference in the pH range 3 to 6. The amounts of ABTS^·+ scavenged by one molecule of AA, AA-2G and 6-Octa-AA-2G after 2 h of reaction at pH 6.0 were approximately 2.0, 3.4 or 3.9 molecules, respectively. This study demonstrates that the quantity of ABTS^·+ quenched by AA-2G and 6-Octa-AA-2G is superior to that of AA in a long-term reaction.

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Gagaminine, a steroidal alkaloid isolated from the roots of Cynanchum wilfordii, exhibited potent inhibitory effects on aldehyde oxidase activity and lipid peroxidation. To determine whether it would be possible to mass produce this active component, which would be useful for animal tests, we tried to synthesize it using in vitro cell culture methods with various growth conditions. In a previous study it was found that calli were easily induced from the stem of this medicinal plant and cultivated effectively on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) 2 mg/l. In this work we attempted to determine the effects of various culture conditions on cell growth and gagaminine synthesis in suspension culture. Gagaminine production was increased markedly when cell growth proceeded to the death phase. Cell growth was more effective with 5% (v/v) sucrose, in the light (at 38 μE/m² · s), on medium containing 2,4-D 2 mg/l, with 2.5 g/10 ml medium as the initial cell concentration. The concentration of gagaminine was optimal with 3% sucrose, in darkness on medium 2,4-D 1 mg/l, with 2.5 g/10 ml medium as an initial cell concentration. However, the highest growth rate was 0.18 d⁻¹, when the gagaminine concentration was seven- and three-fold (at 140 μ/ml) that of the plant stem and 10 ml of medium respectively, on the 50 ml of medium in suspension culture.

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Chitooligosaccharides, the oligomers made up of β-1,4-linked D-glucosamine, are obtained by partial hydrolysis of chitosan, a deacetylation product of chitin. The antioxidant activity of various chitooligosaccharides was tested in vitro with aminoguanidine, pyridoxamine, and Trolox as reference compounds. Hydroxylation of benzoate to salicylate by H₂O₂ in the presence of Cu²⁺ was effectively inhibited by chitobiose, chitotriose, aminoguanidine, pyridoxamine, and Trolox (their IC₅₀ values=18, 80, 85, 10, and 95 μM, respectively), whereas glucosamine and N-acetylchito-oligosaccharides (di-N-acetylchitobiose and tri-N-acetylchitotriose) did not show any inhibitory activity. Chitobiose and chitotriose were more potent than the 3 reference compounds in scavenging hydroxyl radicals produced by photolysis of zinc oxide: IC₅₀ values of the 2 oligomers were 30 and 55 μM, respectively. Such a scavenging activity of these 2 chitooligomers was also shown by the use of another system, a mixture of Fe³⁺/EDTA/ascorbate/H₂O₂, for producing hydroxyl radicals. Only chitobiose and Trolox, of the 10 compounds tested, had the ability to scavenge superoxide radicals generated by a non-enzymatic system using phenazine methosulfate and NADH. Taken together with our unpublished observation that chitobiose and chitotriose are appreciably absorbed from the intestine of rats, the present results suggest that these 2 chitooligosaccharides would act as effective antioxidants in vivo when orally ingested.

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Free radicals react with biological molecules and destroy the structure of cells, which eventually causes free-radical induced disease such as cancer, renal failure, aging, etc. In this study, 6 extracts and 4 pure compounds of Terminalia chebula RETZ. were investigated for anti-lipid peroxidation, anti-superoxide radical formation and free radical scavenging activities. The superoxide radical scavenging of the 4 pure compounds was further evaluated using electron spin resonance (ESR) spectrometry. The results showed that all tested extracts and pure compounds of T. chebula exhibited antioxidant activity at different magnitudes of potency. The antioxidant activity of each pure compound was derived from different pathways and was suggested to be specific.

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In this study, the role of interleukin (IL)-12 on the antimetastatic effect of Z-100 was investigated using wild-type C57BL/6 mice or IL-12p40 knockout (IL-12p40 KO) mice inoculated with highly metastatic B16F10 melanoma. When C57BL/6 mice were inoculated with B16F10 melanoma (2×10⁵ cells/mouse i.v.), Z-100 (10 mg/kg i.p.) significantly suppressed the pulmonary metastasis of B16F10 melanoma 14 d after tumor inoculation. On the other hand, the antimetastatic effect of Z-100 was not observed in IL-12p40 KO mice inoculated with B16F10 melanoma. These results indicate that IL-12 is essentially required for the appearance of the antimetastatic effect of Z-100. Since helper T (Th) 2 cell responses have been reported to have a role in tumor metastasis, the regulatory effect of Z-100 on the immune balance of Th1/Th2 cell responses was investigated. In both C57BL/6 mice and IL-12p40 KO mice bearing B16F10 melanoma, Th1 cytokine production (IL-2, interferon-γ) was significantly suppressed as compared with those in normal mice. On the other hand, Th2 cytokine production (IL-4, IL-10) in these mice was increased. The administration of Z-100 (10 mg/kg i.p.) in C57BL/6 mice bearing B16F10 melanoma improved the balance of Th1/Th2 cell responses from the Th2-dominant state to the normal state. However, the improvement of Th1/Th2 cell responses by Z-100 was not observed in IL-12p40 KO mice bearing the same tumors. In addition, Z-100 significantly increased IL-12 production by macrophages in a concentration-dependent manner, while Z-100 significantly decreased IL-10 production by these cells in vitro. These results suggested that up-regulation of IL-12 production and down-regulation of IL-10 production by Z-100 are related to the improvement of Th1/Th2 cell responses from the Th2-dominant state to the normal state, which resulted in suppression of tumor metastasis.

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The capacities of citrus fruits to inhibit midazolam 1′-hydroxylase activity of cytochrome P450 3A (CYP3A) expressed in human liver microsomes were evaluated. Eight citrus fruits such as ama-natsu, banpeiyu, Dekopon, hassaku, hyuga-natsu, completely matured kinkan (Tamatama), takaoka-buntan and unshu-mikan were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and grapefruit juice (white, Tropicana-Kirin). The addition of a fruit juice prepared from banpeiyu, hassaku, takaoka-buntan or Tamatama caused the inhibition of the microsomal CYP3A activity. The inhibition depended on the amount of a fruit juice added to the incubation mixture (2.5 and 5.0%, v/v). The fruit juice from banpeiyu showed the most potent inhibition of CYP3A. The addition of a banpeiyu juice (5.0%, v/v) resulted in the inhibition of midazolam 1′-hydroxylase activity to about 20% of control without a fruit juice. The elongation of the preincubation period of a fruit juice from banpeiyu (5.0%, v/v) with the microsomal fraction (5 to 15 min) led to the enhancement of the CYP3A inhibition (5% of control). Thus, we discovered ingredients of banpeiyu to be inhibitor(s) or mechanism-based inhibitor(s) of human CYP3A activity, but the inhibitory effects of them were somewhat lower than those of grapefruit.

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